Intermittent warm blood cardioplegia induces the expression of heat shock protein-72 by ischemic myocardial preconditioning

doi:10.1016/S0967-2109(03)00078-4

Cardiovascular Surgery, Vol. 11, No. 5, pp. 367–374, 2003  2003 The International Society for Cardiovascular Surgery Published by Elsevier Ltd. All rights reserved. 0967-2109/03 $30.00

www.elsevier.com/locate/cardiosur

Intermittent warm blood cardioplegia induces the expression of heat shock protein-72 by ischemic myocardial preconditioning M. Chello∗, P. Mastroroberto†, G. Patti§, A. D’Ambrosio§, G. Di Sciascio§ and E. Covino∗ ∗

Interdisciplinary Center for Biomedical Research (CIR), Department of Cardiovascular Sciences, Units of Cardiac Surgery, University Campus Bio-Medico of Rome, Via E. Longoni 83, Rome 00155, Italy, †Department of Clinical and Experimental Medicine, University of Catanzaro, Italy and §Interdisciplinary Center for Biomedical Research (CIR), Department of Cardiovascular Sciences, Units of Cardiology, University Campus Bio-Medico of Rome, Rome 00155, Italy Objective: Recent studies have demonstrated that the induction of heat shock protein-72 (HSP72) by different stimuli preserves the heart function after cardioplegic arrest. Based on these findings, we investigated whether intermittent warm blood cardioplegia would induce changes in the myocardial expression of HSP72. Methods: Forty patients scheduled for aortocoronary bypass were randomly assigned to receive either cold or warm intermittent blood cardioplegia. In all patients HSP72 and HSP72 mRNA were assayed in biopsies from the right atrium at baseline, and during the reperfusion period. Plasma CK-MB and troponin-T, and myocardial oxygen extraction and lactate release were also measured. Results: In both groups, myocardial expression of HSP72 increased throughout the reperfusion period, but the values of HSP72 band lengths were significantly higher in the warm group. Correspondingly, HSP72 mRNA levels increased progressively in both groups, with significant difference between groups observed in biopsies at the reperfusion. Warm blood cardioplegia was associated with lower levels of CK-MB and troponin-T. Myocardial oxygen extraction and lactate release were higher during intermittent warm cardioplegia, indicating a more profound ischemic anaerobic metabolism in the warm group. Conclusions: Intermittent warm blood cardioplegia induces an increased expression of HSP72 and it is associated with a better myocardial protection, by a mechanism involving a variant of the classical ischemic preconditioning model.  2003 The International Society for Cardiovascular Surgery. Published by Elsevier Ltd. All rights reserved. Keywords: Blood cardioplegia, Temperature, Heat shock protein, Preconditioning

Introduction During the past decade the increase in the number of risk factors in patients with coronary artery disease Correspondence to: M. Chello. Tel.: +39-6-2254-1591; fax: +39-62254-1456; e-mail: [email protected]

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referred for coronary artery bypass grafting has paralleled increasingly aggressive attempts to provide maximal myocardial protection during cardioplegic cardiac arrest. In the recent years there has been significant interest in inducing heat shock proteins (HSPs) in the heart and examining their cardio protective capabilities [1–6]. HSP72, the inducible isoform of the HSP 70 family, has been shown to be 367

Intermittent warm blood cardioplegia induces the expression of heat shock protein-72: M. Chello et al.

constitutively expressed in the hearts of patients undergoing cardiac surgery [2,3]. HSPs belong to a family of stress proteins that actively protect the myocyte from not only further heat stress, but other metabolic insults. Many recent studies in a number of different models have demonstrated that different types of stresses, other than hyperthermia, may induce the synthesis of HSP. These stimuli include ischemia and hypoxia [7–10], which are associated to a rapid expression of HSPs in the heart, which continues during cardioplegic storage [11]. Amrani and co-workers [4,5] have demonstrated a protective effect of heat stress on both endothelial and mechanical functions after cardioplegic arrest. Jajakumar and co-workers [12] reported that heat stress caused beneficial changes in high-energy phosphate metabolism in the rat hearts subjected to cardioplegic arrest and ischemia, with a decreased rate of high energy phosphate depletion and increased recovery of ATP and phosphocreatine levels during reperfusion. Intermittent warm blood cardioplegia has been reported to well preserve the myocardium during coronary surgery [13,14], and it has been suggested that the exposure of myocardium to brief period of warm ischemia during cardiac arrest might realize a variant of the heart preconditioning model [15]. Based on these findings, we sought to investigate whether intermittent warm blood cardioplegia would induce changes in the myocardial expression of HSP72 during coronary surgery.

Materials and methods Patients Forty patients scheduled to undergo no urgent aortocoronary bypass grafting at the Medical School of Catanzaro were randomly assigned by hospital number to one of two cardioplegia groups. All patients had angina on effort on admission and were receiving some combination of nitrate vasodilators, calcium-channel and β-blocking agents. The two groups were similar in terms of the mean age, male dominance, preoperative hemodynamic data, and the number of coronary arteries revascularized (Table 1). Patients had good left ventricular function, judged on the basis of a preoperative cardiac angiogram (left ventricular ejection fraction, ⬎0.55), and had no evidence of pulmonary disease, judged on the basis of a chest roentgenogram and lung volume. The same standard anesthesia was used in all patients. After premedication, a Swan-Ganz catheter was positioned into the central pulmonary artery and a radial artery cannula was inserted. Anesthesia was induced with thiopental sodium, and muscle relaxation was achieved with pancuronium; analgesia was provided with fentanyl. The pump (Sarns roller pumps and Dideco hollow fibres oxygenators) was 368

primed with 1500 ml of Ringer’s lactate plus 200 ml of 20% mannitol. The heart was exposed through a median sternotomy, and 300 U/kg of sodium heparin was administered intravenously before cardiopulmonary bypass (CPB) to produce an activated clotting time of greater than 400 s. A coronary sinus catheter was inserted through the right atrium. The hematocrit value was maintained between 20% and 25%, and pump flows were kept between 2.0 and 2.5 l/min/m2 to maintain a mean arterial pressure of between 50 and 70 mmHg. The left ventricle was vented through the aortic root. After decannulation, protamine sulphate (10 mg/ml; Lilly, Inc) was administered intravenously at a dose of 1 mg/300 units of heparin to neutralize the heparin. Cardioplegic groups Cardiac arrest was achieved in all patients by the infusion of a high-potassium (26 mEq/l) blood cardioplegic solution into the aortic root at a rate of 200– 300 ml/min until diastolic arrest was achieved, followed by low potassium cardioplegia (12 mEq/l) repeated after the completion of each distal anastomosis at a rate of 160 ml/min, with intervals not exceeding 18 min (range 12–18 min). The St. Thomas I solution was used in both groups. Blood cardioplegia was prepared by mixing four parts of oxygenated blood with each part of hyperkaliemic crystalloid solution. Blood was collected from the oxygenator using a 1/4 in. tube, mixed with the St. Thomas I solution and was delivered via a separate circuit consisting of a roller pump (Sarns), a reservoir (Terumo), a heat exchanger bubble trap (Medtronics Hall, Minneapolis, MN), and an in-line ultrasonic flow probe. The temperature of this solution was 37 and 5 °C in the warm and cold groups respectively. The body temperature of the patients in the warm blood cardioplegia group was actively warmed to 37 °C during bypass. The myocardial septal temperature in the cold blood cardioplegia group ranged from 10 to 15 °C, and the esophageal temperature in this group was maintained between 25 and 28 °C during the aortic cross clamp period, with rewarming to 37 °C begun during construction of the last distal anastomosis. Reperfusion with warm blood cardioplegic solution before aortic unclamping was never done. Study protocol A wedge resection specimen was excised from the apex of the right atrial appendage immediately before the atrial cannula was introduced. A second and a third specimens were obtained below the purse string suture after aortic cross clamp release and 30 min after cross clamp release. The specimens were immediately frozen in liquid nitrogen and stored in CARDIOVASCULAR SURGERY

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Intermittent warm blood cardioplegia induces the expression of heat shock protein-72: M. Chello et al. Table 1

Demographic and cardiac data

Variable

Warm (N = 20)

Cold (N = 20)

p

Male sex Age (y) NYHA class II NYHA class III Diabetes (non-insulin dependent) Hypertension History of MI Two-vessel disease Three-vessel disease Total CPB time (min) Total cross clamp time (min)

15 (75%) 61 ± 2.1 16 (80%) 4 (20%) 5 (25%) 10 (50%) 15 (75%) 11 (55%) 9 (45%) 91 ± 6.9 61.7 ± 4.7

16 (80%) 59 ± 1.7 17 (85%) 3 (15%) 7 (35%) 8 (40%) 13 (65%) 12 (60%) 8 (40%) 99.4 ± 5.2 57.2 ± 3.3

NS NS NS NS NS NS NS NS NS NS NS

NYHA, New York Heart Association; MI, myocardial infarction; CPB, cardiopulmonary bypass.

it until examination. Arterial blood samples were obtained preoperatively, 1, 4, 12 and 24 h post-operatively for the assay of CK-MB and troponin-T. The samples were collected in heparinized, cooled syringes that were immediately capped and stored in ice until separation and analysis. Finally, for the lactate and oxygen assays, samples were obtained simultaneously from the coronary sinus and the arterial line at the following points: (a) before aortic cross clamping (b) immediately (c) 5 and (d) 15 min after the release of the aortic cross clamp, and (e) at the end of CPB. The study protocol was approved by the ethics committee of the Medical School of Catanzaro. Informed consent was obtained from each patient. Plasma lactate and oxygen consumption Plasma lactate levels was analyzed in duplicate with an automatic analyzer. Blood samples were assayed for the partial pressure of oxygen (PO2) and carbon dioxide, pH, and oxygen saturation (Co-Oxymeter, Instrumentation Laboratory Inc., Lexington, MA). Oxygen content (mmol O2/l) was calculated according to the following formula: 1.39 hemoglobin concentration × oxygen saturation + 0.003 oxygen tension. The arteriovenous difference was calculated as the arterial or cardioplegic content minus the coronary sinus content. Extraction was defined as a significantly positive myocardial arteriovenous concentration difference, as release was defined as a significantly negative myocardial arteriovenous concentration difference. During the cardioplegic arrest the differences in oxygen and lactate contents between the inflow and outflow samples were multiplied by cardioplegic flow. Measurements were made at 37 °C and corrected to the myocardial temperature at the time of sampling. CARDIOVASCULAR SURGERY

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CK-MB and troponin-T assay The serum creatine kinase level and the CK-MB catalytic activity, after inhibition of the MM isoenzyme with monoclonal antibody, were measured by standard methods using a Hitachi 911 analyzer. The serum cardiac troponin-T concentration was analyzed by a commercially available enzyme-linked immunosorbent assay kit (Enzymum Test System ES 22; Boehringer Mannheim, Mannheim, Germany). HSP72 assay Cardiac tissue was immediately frozen in liquid nitrogen. When needed, frozen tissue (10 mg) was homogenized in 400 µl of homogenization buffer (20% sucrose, 125 mM Tris–HCl, pH 6.8, 10% SDS, 120 µg/ml TPCK and TLCK, 100 µg/ml PMSF, 10 µg/ml each of pepstatin, APMSF and leupeptin, and 4 µg/ml aprotinin), centrifuged (2 min, 15,000×g, 4 °C) and the supernatant faction was aliquoted and rapidly frozen in liquid nitrogen. The fractions were maintained at ⫺70 °C until electrophoresis. Samples were prepared for electrophoresis by the addition of DTT to a final concentration of 50 mM, bromophenol blue to a final concentration of 0.01% and then boiled for 2 min. Protein determinations were performed on samples which did not contain DTT with the bicinchoninic acid protein assay method/BCA Protein Assay; Pierce, Rockford, IL) according to the manufacturers directions. Equal amounts of protein were loaded onto a 12.5% acrylamide gel and runned for 2 h at 140 V. The gel was subsequently blotted onto a PVDF membrane (Millipore) overnight at 1 mA and incubated with a monoclonal anti HSP72 antibody (1:1000) dilution. The specific bands were visualized by chemiluminescence (Amersham). The quantitative estimation of the HSP72 was assessed by densito369

Intermittent warm blood cardioplegia induces the expression of heat shock protein-72: M. Chello et al.

metry (NIH Image Program) and expressed as arbitrary units. HSP72 mRNA assay Tissue samples were immediately frozen in liquid nitrogen in the operating room immediately after removal. For northern blots, aliquots of total RNA was fractionated by electrophoresis on a formaldehyde denaturing gel and plotted on nylon membranes. After a prehybridization period of 4–5 h, blots were hybridized overnight with alpha 32P-labeled complementary DNA probes using standard northern blot techniques. After washing, blots were exposed to X-ray films overnight. Autoradiograms generated by northern blots were semi quantified by normalized integrated optical densitometry. Statistical methods Data are expressed as the mean ± the standard error of the mean. A repeated-measures analysis of variance followed by Scheffe´ ’s multiple-comparison analysis was used to test for significant changes over the time course of the study, both within and between groups. An unpaired Student’s t-test was used when appropriate. A p value of less than 0.05 was considered significant.

Results Operative data There were no operative deaths, and no patient sustained a Q-wave myocardial infarction or subendocardial myocardial infarction. In addition, no patients required intraaortic balloon counter pulsation during the post-operative period. The postoperative hemodynamics of the patients in the two groups were comparable (Table 2). During the first post-operative hours, systemic vascular resistance was significantly lower in the warm group (warm, 961.7 ± 69.4 vs. cold 1133.2 ± 53.5 dyn s/cm5, p Table 2

⬍ 0.05) with similar values on the morning of the first post-operative day. Mean infusion rate of vasodilators during the same period were equal. Four patients in the cold group and three in the warm group required dopamine infusion for low cardiac output syndrome (defined as requirement for inotropic support because of a CI of less than 2.1 l/min/m2 and a systolic blood pressure ⬍90 mmHg despite optimization of preload and afterload and correction of any electrolyte The mean infusion rate of vasodilators during the same period was the same. There were no differences between the groups with regard to the total volume administered, the mean urinary output, the need for diuretics, and the total fluid balance during the post-operative period. CK-MB and troponin-T No patient in both groups met the biochemical diagnostic criteria for perioperative myocardial infarction at our institution (peak troponin-T concentration higher than 3.5 µg/l, and peak CK-MB concentration higher than 40 UI/l). There were no statistically significant differences between the warm and cold groups in CK-MK levels at baseline, whereas significantly higher levels (p ⬍ 0.05) were found in the cold group at 1 h (p ⬍ 0.05, 95% CI 0.18–11), 4 h (p ⬍ 0.01, 95% CI 3.1–14), 12 h (p ⬍ 0.01, 95% 3.0–13.9) and 24 h post-operatively (p ⬍ 0.05, 95% 0.6–11.6) (Figure 1, lower panel). There was no significant difference in troponin-T levels between groups in samples taken at baseline and at the end of CPB (Figure 2, upper panel). In both groups, troponin-T levels peaked at 4 h postoperatively, and significantly higher values were found in the cold group at this point (p ⬍ 0.05 vs. warm, 95% CI 0.07–0.7) and 12 h post-operatively (p ⬍ 0.05 vs. warm, 95% CI 0.01–0.6). Lactate release and oxygen extraction Myocardial oxygen extraction is shown in Figure 2 (upper panel). Myocardial oxygen extraction

Post-operative clinical variables

Variable

Cold group

Warm group

p

CI (l/min/m2) Systolic PAP (mmHg) MLAP (mmHg) SVR (dyn s/cm5) LWSWI (gm/m2) 12 h 24 h HR (beats/min) LOA (No)

3.0 ± 0.05 38 ± 5 13.3 ± 0.4 1133.2 ± 53.5

3.2 ± 0.07 35 ± 3.9 14.4 ± 0.6 961.7 ± 69.4

NS NS NS p ⬍ 0.05

25 ± 4 30.1 ± 5.1 81 ± 7.7 4

29 ± 3.6 32.6 ± 4.4 84 ± 9.3 3

NS NS NS NS

CI, cardiac index; PAP, pulmonary artery pressure; MLAP, mean Left atrial pressure; SVR, systemic vascular resistance; LWSWI, left ventricular stroke work index; HR, heart rate; LOS, low out-put state.

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Figure 1 Plasma level of CK-MB (lower panel) and troponin-T (upper panel) in samples from patients of the two cardioplegia groups up to 24 h post-operatively. Base = baseline, 1, 4, 12 and 24 h=1, 4, 12 and 24 h post-operatively. ∗ = p ⬍ 0.05 and ∗∗ = p ⬍ 0.01 cold group vs. warm group

increased similarly in the two groups after cross clamp removal, and only a weakly significant difference between the two groups was observed immediately after the cross clamp release. There were no significant differences in myocardial oxygen extraction between groups at the end of CPB. There was a release of lactate in both groups immediately and 5 min after the release of the aortic cross clamp (Figure 2, lower panel). This was significantly higher in patients of the warm group, at both sampling points (off-cross clamp: p ⬍ 0.01, 95% CI 0.15–0.55; 5⬘ after cross clamp release: p ⬍ 0.05, 95% CI 0.02–0.42). Remarkably, lactate production reverted to lactate extraction 15⬘ after aortic declamping in the cold group, whereas the warm group continued to produce lactate (p ⬍ 0.05, 95% CI 0.05–0.45). At the end of CPB, the release of lactate ceased in all patients of both groups. CARDIOVASCULAR SURGERY

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Figure 2 Lactate production (lower panel) and oxygen extraction (upper panel) before cross clamping (pre xcl), immediately after cross clamp removal (off xcl), 5 (5 xcl) and 15 (15 xcl) min after cross clamp removal, and at the end of cardiopulmonary bypass (end CPB). Lactate production was greater in the warm group, whereas a significant difference in oxygen extraction between groups was observed only after the removal of artic cross clamp (p ⬍ 0.05, 95% CI 0.006–0.22)

HSP72 Heat-shock protein 72 was detectable preoperatively in all patients of both groups. In atrial specimens taken at the second and third sampling points, a significant increase of this stress protein compared with baseline was observed in all patients of the two groups. In both groups, myocardial expression of HSP72 showed a similar pattern of increment from baseline to the end of the cross clamp period (Figure 3, upper panel), reaching the highest observed values at 30 min after cross clamp release (warm: 2.66 ± 0.19 AU; cold 1.95 ± 0.14 AU). However, when data between groups were compared, the repeated-measures analysis of variance showed significant higher increments of HSP72 band length in the warm group in both samples obtained during the reperfusion period (cross clamp release: p ⬍ 0.05, 95% CI ⫺1.00 to ⫺0.06; 30⬘ after cross clamp release: p ⬍ 0.01, 95% CI ⫺1.18 to ⫺0.24) (Figure 4). 371

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HSP72 mRNA HSP72 mRNA was barely detectable preoperatively in patients of both groups. In both cold and warm groups, biopsies taken during the reperfusion period showed a similar pattern of change with a significant progressive increase of HSP72 mRNA over the baseline values (Figure 3, lower panel), with peak values observed 30 min after cross clamp release (warm: 3.2 ± 0.2; cold: 2.46 ± 0.2). Remarkably, in the two samples obtained during the reperfusion period the values in the warm group were significantly higher compared with those observed in the cold group (cross clamp release: p ⬍ 0.05, 95% CI ⫺1.33 to ⫺0.08; 30⬘ after cross clamp release: p ⬍ 0.01, 95% CI ⫺1.34 to ⫺0.13).

Discussion

Figure 3 Upper panel: optical density of immunoblots of HSP72 from right atrial biopsies of all patients in both cardioplegia groups at baseline (base), immediately after cross clamp release (CCR) and 30 min after cross clamp release (30⬘CCR). The values are expressed as arbitrary units. Lower panel: bar graph shows the mean expression of HSP72 mRNA in right atrial biopsies from all patients in both cardioplegia groups. Abbreviations as above

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The results of several recent studies evidence that the myocardial levels of HSP72 may be critical in protecting the heart against ischemia. This has been demonstrated in numerous experimental models of low-flow or regional ischemia using different stress stimuli [4,8,9]. Hutter and co-workers [16] reported that the progressive reduction of the infarct size in rats that were heat-shocked to 42 °C was directly correlated with the amount of HSP72 induced. Using a similar model of ischemia/reperfusion, Gowda and co-workers [7] demonstrated that local heating of the heart was associated with elevated levels of HSP72 and a significant reduction of the infarct size. Finally, Amrani and co-workers showed that in an animal model of hypothermic cardioplegic arrest, the post-ischemic recovery of mechanical and endothelial cell functions significantly correlated with the levels of HSP70 [4,5]. The results of the present study demonstrate that warm blood cardioplegic arrest of the heart followed by normothermic intermittent reperfusion is associated with an increased expression of HSP72 compared with hearts treated with cold blood cardioplegia. In particular, the densitometric analysis of western blots showed increased levels of HSP72 in biopsies from right atria in patients of both groups during the reperfusion period, but the amount of expressed protein was significantly higher in the warm group. A similar pattern of increments for HSP72 mRNA from baseline to the end of the reperfusion period was observed in both groups of patients, with significantly higher values in the warm group. Finally, the increased expression of HSP72 could be responsible for a better myocardial protection during cardioplegic arrest by intermittent infusion of warm blood cardioplegia, as indicated by the reduced CK-MB and troponin-T release in the patients in the warm group. Our findings of an increased myocardial expression of HSP72 after corCARDIOVASCULAR SURGERY

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Figure 4 A representative immunoblot of right atrial biopsies from six patients of the cold group (A–B–C–D–E–F) and six patients from the warm group (G–H–I–L–M–N), taken at the release of aortic cross clamp (panel a) and 30 min after the release of aortic cross clamp (panel b)

onary bypass surgery are in agreement with those of previous studies. Demidov and co-workers [3] reported an elevation of HSP72 and HSC73 in myocardium of patients with coronary disease after surgical revascularization in 40% and 23% of cases, respectively. Also, in the group of patients with the higher content of HSP72, the authors found a significant lower activity of lactate dehydrogenase, creatine phosphokinase and its MB fraction. Perrault and co-workers [17], reported a HSP70 mRNA increase of 1.5–3 fold after bypass, in four patients subjected to brief alternating periods of normothermic ischemia and reperfusion. Taggart and coworkers [2] observed that the amount of myocardial HSP72/HSC73 protein was increased several fold after ischemic insult in four patients operated of CAGB under brief period of normothermic ventricular fibrillation. They also observed an increased expression of HSP72 mRNA in true cut biopsies of left ventricle. On the other hand, the results of our study differ somewhat from those of McGrath and co-workers [1], who did not find HSP induction in right atria of patients after blood cardioplegia. Nevertheless, as already pointed out by other authors [1,3], several methodological problems could account for the differences between previous studies. Problems include the sampling size (only five patients in McGrath report), the type and the temperature of cardioplegic solution (crystalloid or hematic), the length of cross clamp time, mechanical stress during the surgical procedure, collateral flow and blood reperfusion. The lack of any observed increase in the HSP72 content in some cases may be due to different duration of surgical procedure and accordingly, to different time period between the pre-bypass and post-bypass biopsies. In his study, Demidov [3] reported that his post-bypass HSP72 levels were higher in patients with the CPB lasting longer than 2 h. In the specific setting of our study, the findings of CARDIOVASCULAR SURGERY

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an increase expression of HSP72 in atrial samples from patient of the warm group could be somehow difficult to explain. Some aspect, however, could help to clarify the problem. First, the baseline patient characteristics did not differ significantly between the two groups, and both cross clamp time and total CPB duration were also comparable. Moreover, the temperature of perfusing blood in the warm group never exceeded 37 °C, that is significantly below the level of 42 °C reported in literature as the temperature necessary to induce the synthesis of HSP [7,16]. It is therefore possible to speculate that the increase in HSP72 expression observed in the warm group could be the result of an ischemic preconditioning of myocardium following the exposure to brief period of normothermic hypoxia. To this regard, several studies support the idea that a relatively brief period of time after ischemia might be sufficient to elicit a sufficient amount of HSPs in the cardiac tissue. Knowlton and co-workers [18], using an in vivo rabbit model of myocardial ischemia, found that the expression of HSP70 was doubled after 5 min of ischemia, whereas four cycles of 5 min of ischemia alternated to 5 min of reperfusion resulted in a threefold increase in HSP mRNA. In a similar fashion, Marber and co-workers [9] observed a significant increase of myocardial HSP72 contents in rabbit hearts after four 5-min of coronary ligation separated by 10 min of reperfusion. Finally, Valen and coworkers [19] reported an increased expression of HSP72 in atrial samples form patients with unstable angina, and attributed this finding to a preconditioning effect caused by the intermittent ischemia and reperfusion of unstable angina. As currently practiced, intermittent warm blood cardioplegia is associate with periods of normothermic ischemia. In our study, the cardioplegia was delivered in the two groups during 2 min after each anastomosis with a maximal interval of 12–18 min. Cardioplegia was therefore delivered during 17–30% 373

Intermittent warm blood cardioplegia induces the expression of heat shock protein-72: M. Chello et al.

of total duration of aortic cross clamping. In our study, myocardial oxygen consumption and lactate release were greater with warm cardioplegia than with cold cardioplegia, indicating that ischemic anaerobic metabolism became more profound in the warm group. These results are in agreement with those of other studies. In particular, Landymore and co-workers measured oxygen consumption and lactate production during intermittent warm blood cardioplegia [20] and found that lactate production increased in a linear fashion for the duration of the ischemia and oxygen debt occurred in less than 5 min after interrupting the delivery of cardioplegia. The same author also demonstrated in an animal model that preconditioning may be induced when warm blood cardioplegia is delivered intermittently during cardioplegia arrest [15]. Therefore, it may well be that the better cardio protection achieved in the warm group, as indicated by reduced CK-MB and troponin-T release in the patients in the warm group, could be related to a variant of the ischemic preconditioning model. The main limitation of the present study is represented by the fact that changes in HSP72 were studied in the right atria tissue, which differ from ventricular tissue in metabolic and physiologic function [21]. Whether similar changes also occur in human left ventricle is not known. Nevertheless, it should be emphasized that changes in the HSP72 expression occurring in the right atrium cannot necessarily be extrapolated to ventricular tissue.

Conclusions From the results of the present study it seems reasonable to conclude that intermittent warm cardioplegia induces an increased expression of HSP72, which in turn is associated with a reduced myocardial cellular injury as indicated by reduced CK-MB and troponin release. The use of intermittent warm blood cardioplegia represents therefore an attractive potential addition to current methods of myocardial protection.

4.

5. 6. 7. 8.

9.

10.

11. 12.

13. 14. 15. 16.

17. 18. 19.

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Paper accepted 30 April 2003

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