Eur J Cardiothorac Surg 2003;24:932-939
© 2003 Elsevier Science NL
Heat shock protein, inducible nitric oxide synthase and apoptotic markers in the acute phase of human cardiac transplantation
Serban C. Stoicaa,
Duwarakan K. Satchithanandaa,
Carl Atkinsonb,
Susan Charmanc,
Martin Goddardb,
Stephen R. Largea*
a Department of Transplantation, Papworth Hospital, Cambridge, UK
b Department of Pathology, Papworth Hospital, Cambridge, UK
c Department of Statistics, Papworth Hospital, Cambridge, UK
Received 19 April 2003;
received in revised form 30 June 2003;
accepted 3 July 2003.
* Corresponding author. Papworth Hospital, Cambridge, CB3 8RE, UK. Tel.: +44-1480-830541; fax: +44-1480-364334
e-mail: stephenrlarge{at}hotmail.com
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Abstract
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Objective: Solid organ transplantation is associated with activation of apoptotic pathways and other stress markers. We aimed to describe the expression of Bax, Bcl-2, iNOs and Hsp-70 in the endothelium and myocytes of both ventricles and to see if there is any relationship with clinical donor organ failure. Methods: Twelve patients undergoing heart or heart–lung transplantation (including three domino cases) were studied with transmural biopsies from the right (RV) and the left ventricles (LV) at the following points: after donor optimisation; at the end of ischaemic time; and after 10 min of reperfusion. The 1-week endomyocardial RV biopsy was also examined. Five donor hearts turned down purely on functional grounds were analysed also. Results: There was no difference between the RV and the LV for any of the markers at intraoperative assessment. The pattern of expression was not predictive of allograft failure. Donor hearts, however, have a strong pro-apoptotic phenotype, which is largely unopposed by the protective factors Bcl-2 and Hsp-70. Furthermore, the intensity of myocyte staining increases over time for Bax (P<0.001) and iNOs (P=0.02). Domino hearts showed a similar pattern. Compared to usable organs, poorly functioning donor hearts have stronger myocardial staining for Bax (P=0.002) and iNOs (P=0.01). Conclusions: Clinical cardiac transplantation is associated with activation of the Bax and iNOs pathways in both ventricles. The myocardium is affected in time-dependent fashion but this is compatible, to a certain extent, with satisfactory allograft function. Donor hearts turned down on the basis of poor haemodynamic performance have significantly higher expression of Bax and iNOs.
Key Words: Cardiac transplantation • iNOs • Heat shock protein • Apoptosis
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1. Introduction
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The sequence of brain death, ischaemia and reperfusion affects the function of the cardiac allograft in an unpredictable fashion. It is increasingly accepted that brain death has a bigger impact on the right ventricle (RV) and it is RV failure that is more prevalent post-transplant. The endothelium plays an important modulatory role in all these stages [1]. When the ischaemia–reperfusion insult is less than immediately lethal for individual endothelial cells or myocytes they enter specialised survival/apoptosis programmes [2,3]. Ample experimental evidence suggests that modulation of subcellular pathways ameliorates cardiac ischaemia–reperfusion injury in general [4–6] and also in the special case of transplantation [1,7–9]. Donor hearts have an elevation of both TNF-
and IL-6, which is particularly marked in organs with poor left ventricular function [10]. Proinflammatory cytokines expressed at such high levels may account directly for donor heart dysfunction but also for triggering subcellular defence responses. TNF- is able to induce not only apoptosis but also heat shock protein 70 (Hsp-70) and iNOs. The second phase of TNF-mediated contractile impairment is thought to be mediated by nitric oxide (NO) produced by the inducible isoform (iNOs) [5]. Of the many mediators currently scrutinised we focused on Hsp-70, NO, and the pro-/anti-apoptotic pair of proteins Bax/Bcl-2.
Transgenic and molecular techniques have now matured to the extent that they can be used clinically. Extrapolation of bench research to human physiology has occasionally been disappointing in cardiac preservation for transplantation [1]. Knowledge of the sequence in which the above pathways are activated would pave the way for targeted clinical application. A longitudinal study was therefore designed in which donor hearts were serially biopsied from the RV and the left ventricle (LV) in order to characterise the myocardial and endothelial expression of four ‘stress markers’.
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2. Patients and methods
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2.1. Donors and cardiac sampling
Seventeen human hearts were studied with serial transmural biopsies from both ventricles at the following time points:- D, in the donor;
- I, at the end of warm ischaemic time (i.e. end of implantation);
- R, after 10 min of reperfusion;
- 1 week, at 1 week postoperatively (RV only, endomyocardial).
All donors were optimised by the routine use of a hormone cocktail infusion and haemodynamic interventions were guided by Swan-Ganz catheter data. Five hearts without palpable coronary artery disease were turned down on functional grounds based on previously described haemodynamic criteria and the other 12 were transplanted (three as domino). The mean age of the donors was 38.5±11.5 years. The organs studied were implanted as 10 heart (three live donors, see above) and two heart–lung transplants, with an average ischaemic time of 187.2±35.5 min. The mean age of the recipients was 49.4±9.3 years and the transpulmonary gradient was 9.9±2.5 mmHg. The indications for cardiac transplantation were: idiopathic cardiomyopathy (n=6), ischaemic cardiomyopathy (n=2), and valvular disease (n=2). The indications for heart–lung transplantation were cystic fibrosis and secondary pulmonary hypertension, respectively.
Our investigation protocol was approved by the Local Research Ethics Committee. Informed consent was obtained from donor families and from recipients. The retrieval surgeon placed small purse strings on the anterior free wall of the RV and at the LV apex. Serial trucut transmural biopsies were obtained from these sites using a 16G/9-cm Temno needle (Allegiance, IL) and the sutures were tied after the last intraoperative biopsy. The LV site was also used for venting and deairing after implantation. No complications related to this method of tissue sampling were recorded.
2.2. Recipients
All patients had a central venous introducer and a pulmonary artery catheter was immediately floated whenever there was concern about allograft performance. Right heart failure was diagnosed if all the conditions below were satisfied: right atrial pressure >15 mmHg, cardiac index <2.5 l/min per m2, pulmonary capillary wedge pressure <10 mmHg, urine output <1 ml/kg per h and the need for special therapies (one or more of the following: intra-aortic balloon pulsation, pulmonary vasodilators, three or more inotropes). Global graft failure was diagnosed in the presence of borderline haemodynamics (cardiac index <2.5 l/min per m2, wedge pressure >15 mmHg) and the need for special therapies as defined above. Immunosuppression consisted of triple therapy with steroids, cyclosporine and azathioprine.
2.3. Histology and statistics
Postoperative RV endomyocardial biopsies were obtained from the 10 heart transplant patients in standard fashion. Only three patients had evidence of rejection graded as IA according to the ISHLT classification. In two patients with allograft failure the complication was rapidly fatal and the 1-week biopsy was replaced with a post-mortem biopsy obtained within 24 h. Formalin-fixed paraffin-embedded sections were stained for Hsp-70, inducible nitric oxide synthase (iNOs) Bcl-2 and Bax using the Dako Techmate 500 X-Y autostainer (Dako, Glostrup, Denmark), which is based on capillary action. All reagents but the monoclonal antibodies and the phosphate buffered saline (Oxoid, Oxford, UK) are a component part of the Techmate kit (details in Table 1). Experiments with positive controls were performed routinely and negative controls were tested by omission of the primary antibody. Sections were examined independently by two observers blinded to the patient's identity and to the side and timing of the biopsy. Endothelial staining was labelled as present or absent on the arterioles and on non-muscularised vessels, whereas myocyte staining intensity was graded from 0 (absent) to 3. Results are reported as a percentage of cases staining for an individual marker. Comparisons were made with Pearson's chi squared and Fisher's exact tests and statistical significance was attributed when P was less than 0.05.
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3. Results
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3.1. Histological pattern
Haematoxilin-eosin staining showed normal tissue architecture, with no ischaemic injury or indirect evidence of apoptosis. Expression of stress markers in the peritransplant phase is summarised in Table 2. Hsp-70 and Bcl-2 were absent in donor cardiac tissue in biopsies D, I and R, but Bcl-2 appeared occasionally on intravascular and perivascular lymphocytes (Fig. 1A)
. (Hsp-70 was detectable intraoperatively in only two hearts. In one case it was expressed weakly in the endothelium and myocardium of D biopsies but not subsequent to that. In the other patient it was strongly expressed in I and R biopsies.) Hsp-70 was absent on the endothelium but present in all myocytes with variable intensity at 1 week (Fig. 1B). In contrast, iNOs stained both the endothelium and the myocytes in all peri- and postoperative biopsies (Fig. 1C,D). Similarly, Bax stained positive on all myocytes, most capillaries and some arterioles in all biopsies (Fig. 1E,F). Only three hearts stained positive for Bax in D biopsies and they were all from brain-dead patients. The three domino D biopsies did not differ markedly (absent Bcl-2 and Hsp-70, absent Bax on endothelium, positive for iNOs).
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Fig. 1. Histology panel. (a) Bcl-2 expression in a reperfusion biopsy. There is positive staining of activated lymphocytes, demonstrating that the antibody is working. (b) Hsp-70 in a 1-week biopsy showing intense myocardial staining. (c) Intense iNOs staining of endothelium and myocytes in an intraoperative biopsy. (d) Intense iNOs staining of endothelium and myocytes in a 1-week postoperative biopsy. (e) Bax staining of a LV donor biopsy. Notice positive central vessel and granular staining of surrounding myocardium. (f) RV end-ischaemia biopsy showing intense Bax staining of microcapillaries and surrounding myocardium.
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3.2. Trend over time and RV vs. LV
The time-dependent variation was statistically significant for both endothelium and the myocardium. Bax, the marker for which an endothelial variation was seen over time, had a tendency to be increasingly upregulated in muscularised (P=0.06) and non-muscularised vessels (P=0.03) (Fig. 2)
. The intensity of myocyte staining for iNOs increased from grade 2 to 3 during transplantation in both RV and LV (P=0.02) (Fig. 3)
. Similarly, the intensity of myocyte staining for Bax increased from grade 1 to 3 over time in both ventricles (P<0.001) and remained statistically significant when RV 1-week biopsies were included (P=0.004) (Fig. 4)
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Fig. 2. Expression of Bax on RV endothelium. (a) Muscularised vessels, p=0.06. (b) Non-muscularised vessels, p=0.03.
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Fig. 3. Perioperative RV myocyte staining of iNOs. All cases stained positive and data are shown as percentage of cases ranging from 1 (low intensity) to 3 (high intensity). The intraoperative variation includes time points D to R and is statistically significant (p=0.02). The intraoperative LV myocyte staining had an identical pattern (p=0.02). When 1-week results are included the overall variation is not statistically significant (p=0.34).
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Fig. 4. Perioperative RV myocyte staining for Bax. All cases stained positive and data are shown as percentage of cases ranging from 1 (low intensity) to 3 (high intensity). The intraoperative variation includes time points D to R and is statistically significant (p<0.001). The intraoperative LV myocyte staining had an identical pattern (p<0.001). When 1-week results are included the overall variation remained statistically significant (p=0.004).
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3.3. Donor organ failure
Four of the patients had donor organ failure, isolated RV failure in three cases and global failure in one case. There was no relationship between expression of iNOs, Bax and Hsp-70 in the endothelium or the myocardium in biopsies R or 1-week and development of donor organ failure. None of the three domino recipients developed allograft failure.
3.4. Unused donor hearts
Hsp-70 and Bcl-2 were also absent from all unused donor hearts. There was no difference in endothelial staining for Bax between used and unused donors. Compared to transplanted hearts, unused donors had less endothelial staining but more prominent myocyte staining for iNOs (Figs. 5 and 6)
. Similarly, unused donors had significantly more intense Bax staining of myocytes (Fig. 5).
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Fig. 5. Expression of Bax and iNOs in RV myocytes of used (n=10) vs. unused (n=5) donor hearts. (a) Bax, p=0.002. The pattern in the LV was similar (p=0.01). (b) iNOs, p=0.004. The pattern in the LV was similar (p=0.004).
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Fig. 6. Expression of iNOs in RV capillaries in used (n=10) vs. unused donor hearts (n=5), p=0.02. LV data showed a similar pattern (p=0.09).
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4. Discussion
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4.1. Histological pattern and the influence of brain death
Our results show that clinical cardiac transplantation is associated with activation of Bax and iNOs in both ventricles. In broader terms, detection of a given protein by immunohistology gives us little indication about its functional engagement. However, it was not our intention to evaluate the prevalence of apoptosis in clinical transplantation, its contribution to allograft failure or its mechanism: via transmembrane (type I) or mitochondrial (type II) signals. In the quest for a marker of allograft function, we were mainly interested to see what the balance of regulatory apoptotic proteins is. Bax was consistently expressed by myocyte and endothelia beyond the donor biopsy. Bcl-2 neutralises the apoptotic tendencies of Bax [11] but the reasons for the lack of Bcl-2 in our patients are speculative. Perhaps cells adopt a survival program that involves mediators other than Bcl-2. A stepwise downregulation of the antioxidant gene Bcl-2 may also be taking place in the allograft [9]. It is not clear, however, to what extent this mechanism is also active in the human heart for two reasons: first, it is surprising that Bcl-2 is absent even before ischaemia; second, other investigators demonstrated upregulation of caspases in dysfunctional donor hearts, suggesting the possibility of a type I apoptotic mechanism [12]. In a canine experiment of ischaemia and reperfusion Zhao et al. showed a similar imbalance of Bax and Bcl-2 [2]. The fact that such an imbalance is also possible clinically is demonstrated by the overall satisfactory performance of allografts in our study. Depre and Taegtmeyer postulate that a simultaneous perturbation of energy metabolism, contractile function and gene expression is necessary before the cells commit themselves to apoptosis [3]. We analysed elsewhere the time-dependent variation in energy stores and endothelial activation for the human cardiac allograft [13,14]. How the survival program is achieved in the majority of allografts is not known but the endothelium and the myocardium appear to act in concert to preserve function. Myocyte apoptotic stimuli are abundant on both cytokine (e.g. TNF-) and mitochondrial (e.g. free radical) pathways. Programmed cell death is an energy-dependent process, but the levels of high energy phosphates, Bax and Bcl-2 required to progress to apoptosis are not defined. The current study suggests that a marked imbalance in favour of Bax is compatible with allograft survival. Nevertheless, experiments showed that upregulation of anti-apoptotic Bcl-2 is worth exploring as a therapeutic avenue to reduce endothelial activation [11] and enhance allograft function [9]. The salutary activity of Bcl-2 extends beyond its anti-apoptotic effects through inhibition of NF-
B activation [11].
Heat shock proteins are families of highly conserved molecules that afford cytoprotection by intracellular assembly, folding and translocation of oligomeric proteins. Hsp-70 is not constitutively present in the heart, but in heat-shocked rats Hsp-70 is predominantly induced in the endothelium [4]. Using a balloon expansion brain death animal model, Yeh et al. showed that Hsp-70 mRNA is present in cardiac tissue within 4 h after inducing the lethal increase in intracranial pressure [15]. We have not found reports of Hsp-70 immunohistology in the brain dead human donor. In postoperative biopsies from 15 patients (obtained at an unspecified time after transplant), Baba and colleagues showed, similar to our results, that Hsp-70 was generally expressed by the myocytes but not by the endothelium [16]. Perioperative stress was not sufficient for early induction of Hsp-70 in the current study, with expression taking place sometime between reperfusion and 1 week postoperatively.
NO is a potent vasodilating autacoid whose role in transplantation is still debated. Of the three NO synthases, the inducible isoform is cytokine-regulated and not expressed in the normal heart [17]. One study measured mRNA iNOs in 26 donor hearts (including 10 dominos) and found expression in one heart only, from a brain-dead patient [10]. That particular heart, however, did not express TNF-, which is one of several triggers for iNOs transcription. In contrast to that, our study showed that all 10 donors evaluated had iNOs staining in the endothelium and the myocytes. This may be explained by the different methods employed, as there can be less than good correlation between mRNA levels and immunostaining [17]. Expression of iNOs by the three domino donors is difficult to explain. Postoperative cardiac expression of iNOs is, however, in keeping with other studies that reported increases at different time points post-transplantation [17,18]. Together with the elegant experiment of Skarsgard et al. these results suggest that the transplanted heart exhibits a NO-mediated vasodilatation, which probably influences allograft performance and subsequent vasculopathy [19].
It is difficult to draw conclusions about the influence of brain death, since we only had three dominos with a pattern of expression comparable to brain dead donors. Occasionally the expression was surprising (the only donor with Bax myocyte staining higher than grade 1 in both ventricles was a domino, Fig. 4). These observations are perhaps explained by the inflamed intrathoracic milieu of cystic fibrosis, a condition associated with endothelial perturbations. Our conclusions therefore describe an average set of organs used in clinical transplantation.
4.2. Trend over time and RV vs. LV
A gradual increase in the number of cases staining positive for Bax and iNOs was seen (Figs. 2–4), with no difference between the two ventricles. This pattern suggests that the maximal inflammatory insult takes place between immediately after reperfusion and 1 week postoperatively. These data parallel the observations of other groups, which studied apoptosis in a murine model [20]. After ischaemia and reperfusion programmed cell death of the myocytes was preceded by that of the endothelium, the first to be affected being the small coronary vessels. Endothelial apoptosis reached a peak at 60 min after reperfusion. Cardiomyocyte apoptosis was initially taking place in a perivascular location and it assumed a more homogenous distribution at 120 post-reperfusion. Scarabelli's results indicate a potential role for endothelial-released factors, which may act in paracrine fashion to propagate injury to the cardiac parenchyma [20].
4.3. Donor organ failure and implications for rejection
In patients with chronic heart failure there is overexpression of Bcl-2 but not Bax compared to normal hearts [21]. The work of Olivetti is limited in this conclusion by the small number of patients in each subset of the study and by the use of controls where 3 out of 11 cases were brain-dead donors. In contrast to this, we found overexpression of Bax in human donors and no Bcl-2. The inability to correlate expression of stress markers with donor organ performance may be related to the small number of observations or to our relatively crude definition of allograft dysfunction.
The current study reports on the expression of stress markers before the onset of acute or chronic rejection. Others showed that acute rejection is associated in the human allograft with iNOs expression and apoptosis [22]. Inhibition of iNOs in the acutely rejecting murine heart minimised the reduction in left ventricular diastolic filling [7]. In experiments with iNOs-null recipient mice, other groups showed that iNOs increases allograft survival, possibly by stabilising the Weibel–Palade bodies [8]. When iNOs and cardiac performance were analysed in the absence of rejection the results were conflicting. iNOs expression correlates with systolic and diastolic dysfunction in the human allograft [17,23]. All recipients express iNOs mRNA at some stage after transplant, but this is more frequent in the first 6 months [17]. Furthermore, Wildhirt and colleagues examined coronary flow reserve in the first year post-transplant and demonstrated microvascular dysfunction in 30% of recipients in relation to increased iNOs (but not eNOS) mRNA expression [18]. In the murine allograft, however, the vasoplegia observed involves eNOS- and iNOs-based NO production [20]. Whatever the mechanism, the enhanced vasodilatation predisposes to interstitial oedema and ventricular stiffness, with long-term consequences for the allograft. Cardiopulmonary bypass is insufficient to induce iNOs mRNA expression in the human myocardium subjected to ischaemia and reperfusion [24]. The inflammatory load of the transplanted heart may therefore be higher than in conventional cardiac surgery, with iNOs induction starting as early as in the donor.
4.4. Unused donor hearts
Poorly functioning donor hearts have more intense myocyte staining for Bax and iNOs but much less frequent iNOs expression on the endothelium (Figs. 5 and 6). It appears that in some hearts brain death and the ensuing circulatory abnormalities induce expression of iNOs and Bax beyond a threshold which is compatible with satisfactory pump function. A cause and effect relationship is, however, not demonstrated in the current study. Results from two recent experiments should also be noted in this context. Transgenic mice with marked overexpression of iNOs were surprisingly similar to wild type controls in terms of cardiac performance and myocardial energetics [25]. Conversely, genetically iNOs deficient mice were very vulnerable to ischaemia and reperfusion, the mechanism probably being perturbed transcription of NF-B-dependent inflammatory mediators [6]. These two studies suggest that iNOs expression is essential in protection from ischaemia.
In summary, clinical cardiac transplantation is associated with activation of the Bax and iNOs pathways in both ventricles. Myocardial expression gradually increases from the baseline but this is compatible, to a certain extent, with satisfactory allograft function. These findings may assist future studies of organ preservation and prevention of graft vasculopathy.
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Acknowledgments
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Drs Stoica and Satchithananda were funded by the Garfield Weston Research Foundation. We thank Susan Charman for performing the statistical analysis. Jennifer Silk and Mark Southwood provided excellent technical assistance.
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Top
Abstract
1. Introduction
