Preconditioning is defined as the cardioprotective effects

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1 Preconditioning Prevents Myocardial Stunning After Cardiac Transplantation Roderick W. Landymore, MD, Alexander J. Bayes, MD, J. Thomas Murphy, MD, and John H. Fris, RT King Fahad National Guard Hospital, Riyadh, Saudi Arabia Background. Preconditioning has been shown to reduce myocardial stunning after reversible global ischemia. To determine whether preconditioning improves functional recovery after cardiac transplantation, 16 sheep were randomly assigned to a preconditioning protocol or to a control group. Methods. Preconditioning was achieved with 5 minutes of global ischemia followed by 10 minutes of reperfusion. The heart was then arrested with 1 L of crystalloid cardioplegia, explanted, stored in a transport cooler, and then transplanted into recipient sheep. The total ischemia time was 2 hours. Pressure-volume loops were used to calculate preload recruitable stroke work, the maximum elastance, and diastolic compliance. Linear regression analysis was used to determine the preload recruitable stroke work, maximum elastance, and diastolic compliance and end-diastolic volume relationship. The area under the regression curve for preload recruitable stroke work was defined as the preload recruitable stroke work area. Biopsies were taken for high-energy phosphates. Results. Systolic function, represented by preload recruitable stroke work area, was preserved after cardiac transplantation in preconditioned animals. Maximum elastance and diastolic compliance were unaffected by preconditioning or ischemia. High-energy phosphates were better preserved in preconditioned animals. Conclusion. Preconditioning prevented myocardial stunning and preserved high-energy phosphates after experimental cardiac (Ann Thorac Surg 1998;66:1953 7) 1998 by The Society of Thoracic Surgeons Preconditioning is defined as the cardioprotective effects of multiple, brief episodes of myocardial ischemia [1]. Preconditioning has been documented in experimental animal models [1, 2], has been shown to preserve high-energy phosphates in patients who have coronary artery bypass operations [3], might play a role in limiting the extent of myocardial infarction in patients with unstable angina [4], and has been reported to occur during coronary angioplasty [5]. Because preconditioning lessens myocardial dysfunction after reversible global ischemia [6, 7], it is conceivable that preconditioning could reduce myocardial stunning after cardiac Material and Methods Sixteen sheep, weighing between 20 and 25 kg, were randomly assigned to a preconditioning protocol or to a control group. The animals were cared for according to the guidelines set forth by the Canadian Council on Animal Care. Accepted for publication May 28, Presented at the Canadian Cardiovascular Society, Montreal, Quebec, Canada, October 29 November 2, Address reprint requests to Dr Landymore, Department of Cardiac Sciences, King Fahad Hospital, P.O. Box 22490, Riyadh, Saudi Arabia. Surgical Preparation The animals were anesthetized and prepared for cardiopulmonary bypass. The surgical preparation and instrumentation has been described in detail in a previous publication [8]. Hemodynamic Measurements Systolic and diastolic function were determined by simultaneous measurement of left ventricular pressure and volume [9]. A detailed description of the pressurevolume catheters was published previously [8]. Preload recruitable stroke work (PRSW), the maximum elastance (E max ), and diastolic compliance (DC) were automatically calculated from pressure-volume loops with specially designed software. The PRSW was indexed to body weight. Hemodynamic measurements were made during rapid volume loading. The measurements were made before cardiac transplantation and repeated at 90 minutes and 150 minutes after releasing the aortic cross-clamp (Fig 1). Linear regression analysis of PRSW and end-diastolic volume (EDV) was used to determine the PRSW-EDV relationship. Preload recruitable stroke work area (PR- SWA) was defined as the area under the regression curve [10] and was calculated from the following formula: PRSW 1 2 M sw (EDV 50 V sw ) 2, where M sw represents the slope of the regression curve and V sw represents the X intercept. End-diastolic volume was extrapolated to 50 ml (EDV 50 ). Maximum elastance (E max ) was regressed against EDV and extrapolated to an EDV of 50 ml so that the measurements could be compared between groups under a similar loading condition. Diastolic compliance 1998 by The Society of Thoracic Surgeons /98/$19.00 Published by Elsevier Science Inc PII S (98)

2 1954 LANDYMORE ET AL Ann Thorac Surg PRECONDITIONING FOR CARDIAC TRANSPLANTATION 1998;66: Fig 1. Experimental procedure and timeline. The aortic cross-clamp was removed after cold storage and transplantation (2 hours of ischemia). The heart was then allowed to beat for 60 minutes before terminating cardiopulmonary bypass. Hemodynamic measurements were performed at 90 and 150 minutes after releasing the aortic cross-clamp. Myocardial biopsies were taken at 90, 120, and 150 minutes after releasing the aortic cross-clamp. (Con control group; HM B hemodynamic measurements and myocardial biopsies were taken; Pre-C preconditioned animals; Pre-Bypass measurements taken before cardiopulmonary bypass) was regressed against EDV to determine the DC-EDV relationship. DC was extrapolated to an EDV of 50 ml. Myocardial Preservation and Preconditioning Protocols CONTROL GROUP. Seven animals were anesthetized and prepared for the initial hemodynamic measurements. After completing the measurements, cardiopulmonary bypass was maintained for 15 minutes at 37 C. The heart was then arrested with 1 L ofcrystalloid cardioplegia [11], excised, and placed in a sterile transport cooler. The heart was removed from the cooler after 75 minutes of ischemia and then transplanted into a recipient sheep. The aortic cross-clamp was removed after a total ischemia time of 2 hours. The heart was then reperfused on bypass for 1 hour before the animals were weaned from cardiopulmonary bypass. PRECONDITIONED GROUP. Nine sheep were anesthetized and cannulated for the initial hemodynamic measurements. After completing the initial assessment, the animals were placed on bypass at 37 C and then immediately crossclamped. After 5 minutes of global ischemia, the clamp was removed and the heart reperfused for 10 minutes. The heart was then arrested with 1 L of crystalloid cardioplegia, explanted, and placed in a transport cooler. Seventy-five minutes later the heart was transplanted into a recipient sheep. The cross-clamp was removed after a total of 2 hours of ischemia. After 1 hour of reperfusion, the animals were weaned from cardiopulmonary bypass. Surgical Technique The heart transplantation was performed using the technique originally described by Lower and Shumway [12]. Inotropic support was not used in any of the transplant recipients. Blood gases were monitored and maintained within normal limits. High-Energy Phosphates Biopsy specimens were obtained with a Tru-Cut biopsy needle (Travenol Laboratories, Deerfield, IL) before cardiac transplantation, and at 90, 120, and 150 minutes after releasing the aortic cross-clamp (Fig 1). The samples were immediately placed in liquid nitrogen. Highperformance liquid chromatography was used to determine the concentrations of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate [13]. The measurements have been reported as mol/g (dry weight). Statistical Methods The results are reported as the arithmetic mean the standard error of the mean. Analysis of variance was used for the analysis of the hemodynamic data and the Student s t test was used to analyze the measurements of high-energy phosphates. Results Global ischemia during preconditioning resulted in a gradual decrease in heart rate followed by cardiac arrest. After the removal of the aortic cross-clamp, the myocardium became pink, and sinus rhythm resumed spontaneously in all animals. After cardiac transplantation and release of the aortic cross-clamp, ventricular fibrillation occurred spontaneously in both groups. The heart, however, was defibrillated without difficulty in all animals on the first or second attempt and without the need for lidocaine. The animals were weaned from cardiopulmonary bypass without the use of calcium or inotropic support and survived the period of reperfusion until the end of the protocol. Hemoglobin during reperfusion measured g/l in the control group and g/l in the preconditioned animals. Preload Recruitable Stroke Work Area Preload recruitable stroke work area (PRSWA) is illustrated in Table 1. It was similar in both groups before cardiac However, 90 minutes after ischemic arrest, PRSWA was significantly lower in the control

3 Ann Thorac Surg LANDYMORE ET AL 1998;66: PRECONDITIONING FOR CARDIAC TRANSPLANTATION 1955 Table 1. Preload Recruitable Stroke Work Area a Group PRE-B R 2b 90 Min c R 2 p value d 150 Min c R 2 p value d Control Preconditioned a Data are presented as mean standard error of the mean. (joules 10 7 per beat per 100 g). b Correlation coefficient for linear regression analysis. c Post bypass measurements made 90 and 150 minutes after releasing the aortic cross-clamp. d Comparisons between groups using analysis of variance. PRE-B results of preload recruitable stroke work area before cardiopulmonary bypass. group ( p 0.04). In contrast, systolic function was not affected by cold hypothermic storage in the animals that had been preconditioned; PRSWA was somewhat higher in comparison with the pretransplantation assessment. The trend continued to 150 minutes after release of the aortic cross-clamp; PRSWA was significantly higher in the preconditioned animals ( p 0.02) compared with the control group. Not only was PRSWA lower during reperfusion in the control group but it failed to recover during the period of observation. The slight increase in PRSWA observed in the preconditioned group after transplantation did not represent an absolute increase in mechanical function but rather preservation of systolic function, which is often decreased after cold cardioplegic arrest. Maximum Elastance Maximum elastance was similar in both groups before transplantation (Table 2) and was unaffected by transplantation and reperfusion in the control group but tended to be higher in the animals that were preconditioned. Diastolic Compliance As shown in Table 3, DC was not statistically different between groups before cardiac Diastolic compliance remained similar in preconditioned and nonpreconditioned animals after transplantation and was unaffected by 2 hours of ischemia or preconditioning. High-energy Phosphates Adenosine triphosphate and its precursors were relatively unchanged in preconditioned animals (Table 4). The control group, however, demonstrated significant reductions in adenosine triphosphate at 90 minutes ( p 0.03) and 120 minutes ( p 0.04) after cardiac arrest. Table 2. Maximum Elastance of the Left Ventricle a Group PRE-B R 2 90 Min b R Min b R 2 Control Preconditioned a Data are given as mean standard error of the mean (mm Hg/ ml). b Postbypass measurements made at 90 and 150 minutes after releasing the aortic cross-clamp. PRE-B measurements taken before cardiopulmonary bypass and Adenosine diphosphate and adenosine monophosphate were also relatively well preserved in the nonpreconditioned animals after Comment Although ischemic preconditioning was described 10 years ago [1], only recently has it been shown to exert cardioprotective effects after cardiopulmonary bypass and cardioplegic arrest. Alkulaifi and colleagues [3] reported that high-energy phosphates were better preserved and troponin-t release reduced in preconditioned patients who had coronary artery bypass operations, whereas we demonstrated that animals preconditioned during warm blood cardioplegia have better functional recovery when subjected to warm ischemia [8]. Although previous preconditioning protocols described the use of three or four periods of ischemia alternating with reperfusion [1, 2], it is now recognized that a single episode of ischemia, lasting no more than 5 minutes, will precondition the myocardium [14]. The beneficial effects of preconditioning, however, are attenuated when coronary artery occlusion is preceded by 40 to 65 preconditioning cycles [15]. The delay between the preconditioning protocol and the period of coronary artery occlusion might also be a limiting factor. Li and Kloner [16] reported that the preconditioning effect is lost if the delay between preconditioning and the sustained period of ischemia exceeds 60 minutes. Lastly, preconditioning might be ineffective if the duration of coronary artery occlusion exceeds 90 minutes [17]. With these limitations in mind, the preconditioning protocol for this experimental investigation consisted of a single 5-minute period of global ischemia followed by 10 minutes of reperfusion with the total duration of ischemia limited to 2 hours. Table 3. Diastolic Compliance a Group PRE-B R 2 90 Min b R Min b R 2 Control Preconditioned a Data are given as mean standard error of the mean (mm Hg/mL). There were no statistically significant differences between groups at each measurement interval. b Measurements taken 90 and 150 minutes after release of the aortic cross-clamp. PRE-B measurements taken before cardiopulmonary bypass and

4 1956 LANDYMORE ET AL Ann Thorac Surg PRECONDITIONING FOR CARDIAC TRANSPLANTATION 1998;66: Table 4. High-energy Phosphates a Group PRE-B 90 Min b 120 Min b 150 Min b ATP ADP AMP ATP ADP AMP ATP ADP AMP ATP ADP AMP Control c c d Preconditioned a Data are presented as mean standard error of the mean ( mol/g dry weight). b Additional biopsies at 90, 120, and 150 minutes after releasing the aortic cross-clamp. c p 0.03; d p ADP adenosine diphosphate; AMP adenosine monophosphate; ATP adenosine triphosphate; PRE-B measurements taken before cardiopulmonary bypass. Systolic function was assessed in this experimental model of cardiac transplantation by measuring PRSWA. Preload recruitable stroke work was measured during rapid volume loading, and linear regression was used to determine the PRSW-EDV relationship. Systolic function was represented by the area under the regression curve. Because this measurement is independent of preload and heart rate, the relationship is one of the best overall assessments of left ventricular function [10]. In contrast, isolated measurements of E max or the maximum rate of increase in left ventricular pressure (Dp/Dt) might not accurately assess left ventricular function, particularly under conditions of myocardial ischemia [18]. Measurements of E max reported here emphasize the shortcomings of assessing isolated, preload-dependent measurements of left ventricular function, as E max was unaffected by cardiac transplantation, an observation that is highly questionable considering that the myocardium had been subjected to 2 hours of ischemia. Assessment of PRSWA after transplantation demonstrated decreased systolic function in the control group but preserved function in the animals that had been preconditioned. We demonstrated that preconditioning prevents the stunning effect of cold storage and ischemia that invariably is observed after Preconditioning not only preserved systolic function after transplantation but reduced the depletion of adenosine triphosphate and its precursors. Adenosine triphosphate and its precursors were well preserved in the animals that had undergone preconditioning. In contrast, adenosine triphosphate was significantly reduced at 90 and 120 minutes in the control group. The failure of adenosine triphosphate to be regenerated during reperfusion in the control group, despite relatively normal levels of precursors, indicates that hypothermic mitochondrial dysfunction was present in the nonpreconditioned animals after cold cardioplegic arrest [19]. Our observations lend support to earlier observations in isolated rat hearts [14, 20] and patients undergoing coronary artery bypass operations [3]. This investigation used preconditioned hearts for experimental cardiac There have been several studies that examined the effects of preconditioning and myocardial storage on myocardial recovery in isolated heart preparations. Saitoh and colleagues [21] recently reported that rat hearts preconditioned with two 2.5-minute episodes of global ischemia separated by 10 minutes of reperfusion demonstrated improved functional and metabolic recovery after 8 to 12 hours of storage in University of Wisconsin solution. Valen and associates [22], using a Lagendorff model, preconditioned rat hearts with two 3-minute episodes of ischemia followed by 5 minutes of reperfusion. Diastolic function was better preserved in the preconditioned hearts subjected to warm ischemia but not in hearts stored for 3 to 3.5 hours between 6 C and 8 C. In contrast, Ogino and colleagues [23] have shown that 5 minutes of global ischemia and 10 minutes of reperfusion, preceding 6 hours of cold (4 C) storage, preserved diastolic function. Lastly, Karck and colleagues [24] subjected isolated, working rat hearts to 5 minutes of global ischemia followed by 5 minutes of reperfusion. The hearts were then stored at 4 C for 10 hours in five preservation solutions. There was very little recovery in the control groups, whereas systolic function was preserved in the preconditioned hearts. Our data showed that preconditioning prevents myocardial stunning in an experimental model of cardiac transplantation when the heart is subjected to 2 hours of ischemia. Longer ischemia times might be more relevant to the clinical situation because increased tolerance to ischemia would extend safe transport time and increase the donor pool. However, there was no indication of systolic dysfunction after transplantation, and in a limited number of observations in our laboratory, this effect was maintained for up to 6 hours. Furthermore, recent reports based on isolated rat heart preparations suggest that preconditioning is effective not only under conditions of hypothermia but that the preconditioning effect might be evident after 12 hours of cold storage. Finally, the application of cardiopulmonary bypass during donor retrieval might not lend itself to clinical cardiac Therefore, a different technique of supporting the myocardium during ischemia and reperfusion might have to be designed or the heart might require preconditioning with adenosine, as previously described. References 1. Murray C, Jennings R, Reimer K. Preconditioning with ischemia: a delay in lethal injury in ischemic myocardium. Circulation 1986;74: Schott R, Rohmann S, Braun E, Schaper W. Ischemic preconditioning reduces infarct size in swine myocardium. Cardiovasc Res 1990;66: Alkulaifi A, Yellon D, Pugsley W. Preconditioning the human heart. Ann R Coll Surg Engl 1997;79:49 54.

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