On-pump versus off-pump coronary artery bypass grafting: more heat-shock protein 70 is released after on-pump surgery

[김기환/강원대]

On-pump versus off-pump coronary artery bypass grafting: more heat-shock protein 70 is released after on-pump surgery

Brit Dybdahl^{, , a, b}, Alexander Wahba^c, Rune Haaverstad^c, Idar Kirkeby-Garstad^c, Peter Kierulf^b, Terje Espevik^a and Anders Sundan^a

^a Faculty of Medicine, Institute of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7489, Trondheim, Norway

^b Department of Clinical Chemistry, Ulleval‎ University Hospital, N-0407, Oslo, Norway

^c St Elisabeth Heart Center, Trondheim University Hospital, N-7018, Trondheim, Norway

Received 23 October 2003; 

Revised 23 February 2004; 

accepted 2 March 2004. 

Available online 2 April 2004.

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Abstract

Objectives: The use of cardiopulmonary bypass in coronary artery bypass grafting (CABG) may contribute to the postoperative inflammatory response. The molecular chaperone heat-shock protein (HSP) 70 may be induced by ischemia, and has been detected both in the myocardium and in the circulation after CABG. In vitro, extracellular HSP70 may activate both innate and adaptive immunity. Hypothesizing that use of cardiopulmonary bypass (CPB) leads to more circulating HSP70, we explored the release of it in 10 patients undergoing CABG with the use of CPB, and in 10 patients undergoing off-pump surgery CABG (OPCAB). Methods: Blood samples were taken preoperatively, twice peroperatively, 2 and 6 h postoperatively and the next day. Serum analyses were performed by means of immunoassays. Results: We detected a significant difference in postoperative circulating HSP70 between on-pump and off-pump patients (median peaks of 2849 and 756 pg/ml, respectively, P<0.01, 2 h postoperatively). Interleukin-6 and -8 increased in all patients, without significant differences between the groups. Serum interleukin-10 increased at the end of the operation in 7 of 10 patients operated with cardiopulmonary bypass (median 51.7 pg/ml), but in none of the off-pump patients. Furthermore, in the first group, interleukin-10 correlated with the HSP70 concentration at the end of the operation, r=0.75, P<0.05. Serum markers of myocardial damage were higher in conventional than off-pump patients on day 1 postoperatively: median cardiac Troponin T was 0.358 and 0.126 μg/l, respectively, P<0.01. Correspondingly, median creatine kinase-MB was 23.6 and 7.8 μg/l in on-pump and off-pump patients, respectively, P<0.01. Peak HSP70 correlated with both Troponin T and creatine kinase-MB measured on day 1. Conclusions: Significantly more HSP70 was released into the circulation following conventional than following off-pump CABG. Circulating HSP70 may indicate cellular stress or damage. Furthermore, HSPs are suggested as immunoregulatory agents, and may be important in the host defence postoperatively.

Author Keywords: Cell-signaling proteins; Coronary artery bypass surgery; Heat-shock proteins; Immunology; Ischemia/reperfusion; Off-pump

Abbreviations: ACC, aortic cross-clamp; CABG, coronary artery bypass grafting; CD, cluster of differentiation molecule; CK-MB, creatine kinase-MB; CPB, cardiopulmonary bypass; CRP, C-reactive protein; cTnT, cardiac Troponin T; ECG, electrocardiogram; ELISA, enzyme-linked immunosorbent assay; HSP, heat-shock protein; ICU, intensive care unit; IL, interleukin; IRAK, IL-1R-associated kinase; LVEF, left ventricular ejection fraction; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor-kappa B; NO, nitric oxide; NSAID, non-steroidal anti-inflammatory drug; OPCAB, off-pump coronary artery bypass grafting; PRBC, packed red blood cells; Th, T helper cell; TLR, toll-like receptor; TNFα, tumor necrosis factor-alpha

Article Outline

1. Introduction

2. Patients and methods

2.1. Patients

2.2. Anesthesia and surgical procedure

2.3. Blood sampling

2.4. Serum and plasma analyses

2.5. Statistical analyses

3. Results

3.1. Patients

3.2. Serum HSP70

3.3. Interleukins and serum free hemoglobin

3.4. Markers of myocardial ischemia

3.5. Coronary sinus

4. Discussion

Acknowledgements

References

1. Introduction

Coronary artery bypass grafting (CABG) is conventionally performed with the use of cardiopulmonary bypass (CPB) and cardiac arrest. This method results in high quality anastomoses and excellent clinical results [1]. Nevertheless, complications may be associated with the systemic inflammatory response following CABG [2 and 3]. The inflammatory response has been related to anesthesia, the surgical trauma per se, cardioplegia, ischemia-reperfusion and use of the heart–lung machine. Thus, the increasing number of CABG without CPB (off-pump operations, OPCAB) [4], has renewed the scientific interest in the systemic inflammatory response following heart surgery. Recent publications on randomized studies comparing on- and off-pump procedures, showed comparable cardiac outcome [5 and 6], and significantly lowered in-hospital morbidity without compromising outcome in OPCAB patients [7]. In addition, reduced cytokine responses and less myocardial injury are observed in patients undergoing OPCAB compared to CABG with CPB [8]. (For simplicity, the abbreviation CABG is referring only to operations with CPB in the following text.)

Heat-shock proteins (HSPs) are abundant, soluble, intracellular proteins, normally present neither in blood nor other body fluids [9]. HSPs may be induced under stressful conditions like ischemia-reperfusion, and may then be released into the circulation from necrotic or stressed viable cells. The primary intracellular function of HSPs is as molecular chaperones, counteracting the unfolding, misfolding and pathological modification of critical proteins in e.g. ischemic injury [10].

Extracellular HSPs are suggested as danger signals that lead to the activation of innate and adaptive proinflammatory responses [9]. In vitro, it has previously been shown that monocytes stimulated with the inducible HSP70 release proinflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)α [11 and 12]; and the HSP70-induced signaling may be mediated through the MyD88/IRAK/NF-κB signal transduction pathway [13]. Both Toll-like receptor (TLR)-2 and TLR-4 together with CD14 have been suggested to be involved in this signal transduction [11 and 13].

We have previously shown that the stress-inducible HSP70 (also identified as HSP72) is rapidly released into the circulation following CABG [11]. Furthermore, HSP70 has been detected in atrial biopsies from patients undergoing CABG [14]. We were curious to find a possible difference in the inflammatory response, represented by the release of HSP70, following OPCAB compared to CABG. Considering ischemia induces HSP70, we hypothesized that use of cardiac arrest and CPB leads to increased release of HSP70. Accordingly, the first aim of the present study was to compare HSP70 release, inflammatory response, and markers of myocardial damage in CABG and OPCAB. Secondly, we wanted to explore the kinetics of HSP70 release during and after open heart surgery. The third aim was to eval‎uate the myocardial contribution to circulating HSP70.

2. Patients and methods

This study conforms to the principles outlined in the Declaration of Helsinki.

2.1. Patients

From January to August 2002, 20 consecutive patients referred for elective CABG or OPCAB at the St Elisabeth Heart Center, Trondheim, Norway, were recruited to this prospective study. Ten patients underwent CABG, and 10 underwent OPCAB. The patients had angiographically verified coronary artery disease, were <75 years old and had no signs of infection preoperatively. Patients undergoing reoperations, combined procedures or emergency surgery were not included. The regional ethics committee approved the study, and informed written consent was obtained from each patient.

2.2. Anesthesia and surgical procedure

All patients underwent a procedure with median sternotomy. Anesthesia was induced with diazepam, fentanyl, thiopental, and pancuronium and maintained with isoflurane, fentanyl and nitrous oxide. Cephalothin was used as perioperative antibiotic prophylaxis. An initial dose of 300 IU/kg heparin was given to all patients, and additional heparin was given when necessary to maintain clotting time >480 s during CABG and >350 s during OPCAB. Finally, heparin was neutralized with protamine sulfate. The patients undergoing CABG were cooled down to 34 °C; we used St Thomas cardioplegic solution No. I [15], or cold blood cardioplegia. CPB was performed with non-pulsatile flow of 2.4 l/min per m² body surface area with a roller pump. A membrane oxygenator with synthetic, biocompatible surfaces (Maxima, Medtronic) was used in all CABG operations. The pump prime consisted of 1800 ml Ringer's solution and 7500 IU heparin. The Medtronic Octopus System (Medtronic Inc.) was used to expose and stabilize the target coronary artery during off-pump procedures. All the patients received a trans-atrial retrograde cardioplegia catheter (Edwards Lifesciences Corp.) in the coronary sinus at the beginning of the procedure, which was withdrawn before closure. Correct positioning of the catheter with the tip at the level of the posterior interventricular vein was assessed by palpation of the cannula and transesophageal echocardiography. Furthermore, blood was withdrawn slowly to avoid aspiration of right atrial blood.

2.3. Blood sampling

Blood samples were taken through an indwelling radial artery catheter before induction of anesthesia, before starting CPB (or the corresponding time point for off-pump operations), at the end of CPB (immediately following injection of protamine sulfate), 2 and 6 h thereafter and at 8.00 a.m. the next day. Peroperatively, blood was also collected from the coronary sinus through the retrograde cardioplegia catheter. Plasma samples were centrifuged immediately after collection, whereas serum samples were centrifuged following 30 min on the bench. Both serum and plasma were aliquoted and frozen in several vials at −80 °C until analyzed.

2.4. Serum and plasma analyses

Serum HSP70 was measured with StressXpress Hsp70 ELISA Kit, sensitivity 200 pg/ml (Stressgen Biotechnologies Corp). IL-6 and IL-8 in serum were measured with Quantikine Human Immunoassays (R&D Systems Inc), sensitivities 0.70 and 10 pg/ml, respectively. Serum IL-10 was analyzed with Biosource IL-10 EASIA Kit (Biosource Europe SA), sensitivity 1 pg/ml. Cardiac Troponin T (cTnT) and creatine kinase-muscle brain isoenzyme (CK-MB) were measured by the Department of Clinical Chemistry, Trondheim University Hospital, on an Elecsys 2010 Analyser (Roche), detection limits 0.010 and 0.10 μg/l, respectively. Serum-free hemoglobin was analyzed by means of a photometrical method in the same laboratory. We used the hospital routine analyses for quantification of C-reactive protein (CRP; detection limit 5 mg/l), whole blood hemoglobin concentration, leukocyte and platelet counts.

2.5. Statistical analyses

Because of non-Gaussian distribution of most data, we used non-parametric tests and median values with quartiles in the presentation. Comparisons within groups over time were made by means of the Friedman test and Dunn's multiple comparison test. To compare groups, the Mann–Whitney test was used. We performed Wilcoxon signed ranks test to compare blood samples collected from the coronary sinus and the arterial line at the same time point. Differences were considered statistically significant at the level of P<0.05. For correlation analyses, we used Spearman's rho.

3. Results

3.1. Patients

Preoperative patient characteristics are presented in Table 1; Table 2 shows perioperative characteristics and early clinical outcome. Complete revascularization was achieved in all patients. An independent cardiologist examined serial electrocardiograms (ECGs) from the first postoperative day. No signs of ischemia or acute myocardial infarctions were detected. However, two patients had non-Q-wave myocardial infarctions within 2 days postoperatively, whereas one patient had a minor myocardial infarction 15 days after surgery. Furthermore, two patients had uncomplicated superficial chest wound and/or leg wound infections following vein graft harvesting, and one patient had a more long-lasting deep infection of the leg wound. A fourth patient was treated with antibiotics due to suspected infection with unknown focus. Table 3 presents laboratory data. The whole blood hemoglobin concentration, the first postoperative day, was significantly higher in OPCAB than in CABG patients.

Table 1. Baseline patient characteristics

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Age and LVEF values are median values, with quartiles in parentheses; the rest of the values are given as number of patients. LVEF, left ventricular ejection fraction; NSAIDs, non-steroidal anti-inflammatory drugs.

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Table 2. Perioperative patient characteristics and early clinical outcomes within 30 days of surgery

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Data are presented as medians, with quartiles in parentheses, or number of patients. ACC, aortic cross-clamp; CPB, cardiopulmonary bypass; ICU, intensive care unit; PRBC, packed red blood cells.

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Table 3. Laboratory data

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Median values, with quartiles in parentheses, are given. CRP, C-reactive protein.

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3.2. Serum HSP70

The results of the HSP70 measurements are shown in Fig. 1 and Table 4. The median HSP70 concentrations in the group of CABG patients measured preoperatively, at the start and end of CPB, 2 and 6 h thereafter and day 1 postoperatively, were 288 (0–399), 472 (282–635), 2736 (755–4945), 2849 (1333–6555), 2078 (1156–5567) and 486 (277–655) pg/ml, respectively (interquartile ranges in parentheses). At corresponding time points, median serum HSP70 values in OPCAB patients were 343 (210–439), 396 (165–615), 580 (359–781), 756 (509–1540), 677 (475–1289) and 528 (212–807) pg/ml, respectively. Both groups had a significant increase in their serum HSP70 values 2 and 6 h postoperatively, P<0.001. In the CABG patients, the median HSP70 was significantly increased compared to preoperative levels also at the end of the operation, P<0.01. There were significant differences, P<0.01 between the two patient groups both at the end of the operation, and at 2 and 6 h thereafter. Thus, the total amounts of HSP70 measured in serum (area under the curve) were significantly different in the two patient groups (Table 4).

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Fig. 1. Serum HSP70 values in CABG patients (crosshatched boxes, n=10) and OPCAB patients (white boxes, n=10). The boxes extend from the 25th to the 75th percentile with horizontal lines at the median values. Whiskers show the highest and the lowest values. The median concentrations in the two groups were significantly different, P<0.01, at the end of the operation, and 2 and 6 h thereafter.

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Table 4. Area under the curve

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Serum HSP70, IL-6 and IL-8. Values are given as area under the curve (h×pg/ml), median and interquartile range. HSP70, heat-shock protein 70; IL-6 and -8, interleukin-6 and -8.

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3.3. Interleukins and serum free hemoglobin

We could not detect statistically significant differences in IL-6 concentrations between the two groups at any time point (Fig. 2A, Table 4). Both groups had a significant increase in serum IL-6 2 h postoperatively to median 106.6 pg/ml, interquartile range 61.2–139.8 pg/ml in CABG patients, and 91.4 pg/ml interquartile range 74.9–149.4 pg/ml, in OPCAB patients. This increase was persistent at later time points up to the day after the operation. Serum IL-8 increased moderately during the operation day for both groups, and significant differences between the groups were not observed (Fig. 2B, Table 4). Two hours postoperatively, the median serum IL-8 was 31.3 and 25.5 pg/ml in CABG and OPCAB patients, respectively. IL-10 was not detected in sera from OPCAB patients at all, whereas 7 of 10 CABG patients had IL-10 in serum at the end of the operation; median value 51.7 pg/ml, interquartile range 6.8–134.5 pg/ml (Fig. 2C). Interestingly, these patients had the highest HSP70 concentrations. Serum IL-10 in the CABG group correlated with the HSP70 concentration measured at the end of the operation, r=0.75, P<0.05. Serum free hemoglobin (Hb) was measured to quantify hemolysis due to the use of CPB (Fig. 3). It correlated with HSP70 at the end of the operation and 2 h later, r=0.82, P<0.01 in CABG patients. At 6 h postoperatively, there was no significant correlation between HSP70 and serum free Hb. IL-10 in the CABG group also correlated with serum and free Hb at the end of the operation, r=0.72, P<0.05.

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Fig. 2. (A) Median serum IL-6 and (B) IL-8 concentrations in CABG patients (stippled line, n=10) and OPCAB patients (continuous line, n=10). No significant differences between groups were detected. At the start of the operation, median serum IL-8 values in both groups were below the detection limit (10 pg/ml), but were, for simplicity, set to 10 pg/ml in the figure. (C) Individual values and median (horizontal bar) serum IL-10 concentrations in CABG patients. We could not detect IL-10 in OPCAB patients at any time point.

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Fig. 3. Serum free hemoglobin (Hb) concentrations in patient arterial blood, median values. CABG patients (stippled line, n=10) have significantly higher concentrations than OPCAB patients (continuous line, n=10) at the end of surgery, 2 (P<0.01) and 6 h later (P<0.05).

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3.4. Markers of myocardial ischemia

Preoperative cTnT and CK-MB in the two groups were not significantly different (Fig. 4). cTnT reached peak value on day 1 postoperatively; median 0.358 μg/l, interquartile range 0.209–0.526 μg/l in CABG patients, and median 0.126 μg/l, interquartile range 0.066–0.218 μg/l in OPCAB patients, P<0.01. The median CK-MB on day 1 was 23.6 and 7.8 μg/l in CABG and OPCAB patients, respectively, P<0.01. The peak HSP70 concentration correlated with cTnT, r=0.55, P<0.05, and CK-MB, r=0.56, P<0.05, n=20, on day 1 postoperatively. Furthermore, the peak HSP70 was strongly correlated with aortic cross-clamp (ACC) time, r=0.83, P<0.01 and CPB time, r=0.74, P<0.05, n=10.

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Fig. 4. (A) Serum cardiac Troponin T (cTnT) and (B) creatine kinase-MB (CK-MB) before and after CABG (crosshatched boxes, n=10) or OPCAB (white boxes, n=10). The boxes extend from the 25th to the 75th percentile with horizontal lines at the median values. Whiskers show the highest and the lowest values. Median cTnT and CK-MB concentrations were significantly higher, P<0.01, in CABG compared with OPCAB patients on day 1 after the operation.

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3.5. Coronary sinus

To explore a putative local synthesis in the heart, we measured HSP70, IL-6, IL-8 and IL-10 in blood sampled from both the coronary sinus and the peripheral arterial line at identical time points twice peroperatively. There were no significant differences in HSP70, IL-8 or IL-10 concentrations. The IL-6 quantity, however, was significantly higher, P<0.05 in blood from the coronary sinus than in peripheral arterial blood at the end of the operation, suggesting a local myocardial synthesis of IL-6.

4. Discussion

The main finding in the present study, is the huge difference in serum HSP70 following CABG compared with OPCAB. The HSP70 concentrations increased significantly from preoperative values in both patient groups and reached peak concentration 2 h postoperatively. The median value in CABG patients at that time point, however, was almost 4 times the HSP70 concentration in OPCAB patients. The release of HSP70 after CABG is in accordance with our previously published findings [11].

Several studies have shown that ischemia-reperfusion induces HSP70 in myocardial cells [14 and 16]. In this study, the HSP70 concentration in blood from the coronary sinus did not differ significantly from HSP70 in peripheral arterial blood. We cannot exclude the possibility of a minor contamination by right atrial blood in coronary sinus samples, which might mask eventual differences in HSP70 concentration between the systemic and the coronary circulation. It appears more likely, however, that most of the HSP70 induced in the heart is not released into the systemic circulation, but stays localized in the heart.

Blood cells are another putative source of circulating HSP70 following CABG. HSP70 has previously been detected in monocytes and granulocytes in humans [17], and in immature red cells of chicken embryos [18]. Physical damage of the cells circulating through the heart–lung machine may lead to induction and release of HSP70. The correlation between serum free Hb and HSP70 in CABG patients led us to examine the HSP70 content of erythrocytes from healthy people. The HSP70 content of in vitro lysed erythrocytes diluted in autologous serum was very low (data not shown). As such, erythrocytes appear not to be an important source of HSP70. However, the degree of ex vivo hemolysis in CABG patients may indicate the degree of leukocyte damage; monocytes and granulocytes may be substantial contributors to circulating HSP70 in CABG patients.

Our OPCAB patients had a small but significant increase in serum HSP70. This could be due to the surgical trauma, or the local ischemia caused by the clamping of coronary arteries. In addition, manipulation and stretching of the heart during the surgery may cause increase of HSP70 in the heart. It has been shown in rabbit hearts, that stretch or reduced shortening may lead to induction of HSP70 mRNA [19]. This mechanism may also affect CABG patients, perhaps even more than OPCAB patients; aortic cross-clamping may cause such stretch [19], and we found a strong correlation between ACC time and HSP70 concentration. A long ACC time may indicate more myocardial stretch and more ischemia. Thus, it is tempting to suggest that the heart contributes to the circulating HSP70. The length of ACC, however, also indicates the complexity of the operation, and as such the strength of the stimulus for HSP70 release, whatever the source. On the other hand, patients operated with CPB had more circulating cTnT and CK-MB postoperatively than patients operated off-pump, as shown by others [20]. We detected a correlation between the HSP70 concentration and both cTnT and CK-MB, and the possibility that HSP70 may be an additional marker of myocardial damage cannot be excluded.

Randomization of this study was not carried out. The patients were selected for CABG or OPCAB according to the hospital treatment policy at the time; in routine cases 3-vessel diseased patients were scheduled for standard CABG, and patients with 1 to 2-vessel disease were treated according to an OPCAB protocol. As such, the CABG patients had more distal anastomoses than the OPCAB patients. In addition, the heparin dosages were slightly higher in CABG patients (data not shown). Although we cannot exclude an effect of heparin upon the Hsp70 measurements, the small dosage difference may, most likely, be of minor importance. There was no difference in total operation time between the groups. In our view, the main difference between the patients during surgery apparently was the use of cardiac arrest and CPB in CABG patients, but not in OPCAB patients. Thus, although the lack of randomization is a formal weakness of the study, we suggest that this has negligible influence on the main issue of this investigation.

The systemic inflammatory response in the study patients was evidenced by the increase of interleukins and CRP. Serum IL-6 did not differ significantly between the two groups, but there was a trend of IL-6 being higher in OPCAB patients on day 1 postoperatively. The similarity in IL-6 increases between CABG and OPCAB patients has been described previously [2 and 21]. IL-6 is possibly more related to the surgical trauma than to ischemia. Furthermore, the IL-6 concentration was higher in the coronary sinus than in peripheral blood, supporting previous reports that suggest the myocardium as an important source of IL-6 [22]. We did not detect statistically significant differences in serum IL-8 between the groups, although others have found higher IL-8 concentrations in patients undergoing procedures with CPB [21].

There was a remarkable difference in IL-10 between the groups. This anti-inflammatory cytokine was evident at the end of surgery in 7 of 10 CABG patients, but in no OPCAB patients. Similar results have been described previously [21]. Most studies show that IL-10 suppresses pro-inflammatory cytokines, and serum IL-10 may indicate the magnitude of inflammatory stress. Why patients with release of IL-10 have the highest HSP70 concentrations is, as yet, unknown. Whereas extracellular HSP72 has been shown to induce IL-6, TNF-α and IL-12 in monocytes, IL-10 has not been found in supernatants of monocytes stimulated with HSP72 [23]. It has been suggested, however, that HSP70 induces Th2 type CD4⁺ T cells, producing IL-10 among others [24]. As such, HSP70 may be part of an immunoregulatory response that has the potential to control proinflammatory responses [24].

There is increasing evidence for activation of the immune system by extracellular HSP70 [9, 11, 12, 13, 23 and 24]. Recently, however, questions have been asked regarding the mechanism by which Hsp70 activates innate immunity. Endotoxin contamination of recombinant human Hsp70 preparations has been a problem in previous in vitro studies [25]. Thus, further research both in vitro, and in larger clinical studies is needed in order to understand the clinical implications of Hsp70 release following e.g. heart surgery.

To summarize the present study, we have shown that significantly more HSP70 is released after CABG than after OPCAB, possibly indicating a difference in inflammatory responses, cellular stress or damage, between these two procedures. Furthermore, the release of HSP70 appears rapidly, with peak HSP70 concentration at 2 h postoperatively. Both leukocytes and the heart may contribute to the circulating Hsp70 following open heart surgery. The role of extracellular HSP70 as an immunoregulatory agent may be of importance in the host defence postoperatively.

Acknowledgements

This work was carried out with financial support from the Commission of the European Communities specific research, technological development, and demonstration (RTD) program ‘Quality of Life and Management of Living Resources’, Grant QLK2-2000-336, HOSPATH. We are grateful to Arve Tromsdal for careful examination of all ECGs. Furthermore, we had excellent technical assistance from Marthe Aune, Berit Brusletto and Liv Ryan. During collection of blood samples, we appreciated the encouraging attitude and help from the staff at the St Elisabeth Heart Center.

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