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Ann Thorac Surg 1997;63:147-152
© 1997 The Society of Thoracic Surgeons
Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado
Accepted for publication August 1, 1996.
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. Isolated human right atrial trabeculae were suspended in an organ bath with oxygenated Tyrode's solution at 37°C and field stimulated at 1 Hz. Developed force was recorded. Trabeculae (Warm I/R) received normoxic perfusion before 45 minutes of normothermic simulated ischemia (hypoxic, substrate-free buffer with pacing at 3 Hz) and 120 minutes of reperfusion. Preconditioned trabeculae (Warm IPC) were subjected to 5 minutes of normothermic simulated ischemia and 10 minutes of perfusion before normothermic simulated ischemia-reperfusion injury. Trabeculae (Cold I/R) were subjected to hypothermic (4°C) ischemia (hypoxic buffer) for 4 hours and 60 minutes of reperfusion (37°C). Preconditioned trabeculae (Cold IPC) were pretreated with 5 minutes of normothermic simulated ischemia before hypothermic ischemia and 60 minutes of reperfusion. At the end of reperfusion, trabeculae were frozen at -70°C and assayed for tissue creatine kinase activity.
Results. At the end of reperfusion, warm preconditioned trabeculae (Warm IPC) recovered 51% ± 5% of baseline developed force, whereas warm I/R trabeculae recovered 24% ± 3% (p < 0.05). Tissue creatine kinase levels reflecting preserved tissue viability were sustained in Warm IPC trabeculae (1,183 ± 204 U/g), whereas nonpreconditioned control trabeculae (Warm I/R) exhibited lower levels of enzymatic activity (403 ± 32 U/g) (p < 0.05). In contrast, Cold IPC trabeculae recovered 47% ± 5% and Cold I/R, 56% ± 8% of baseline developed force at the end of reperfusion (p > 0.05).
Conclusions. We conclude that transient ischemic preconditioning protects human myocardium against normothermic ischemic injury.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Tolerance of a myocardial ischemia-reperfusion injury can be induced by a preceding transient episode of cardiac ischemia. This potent form of cardioadaptation is termed ischemic preconditioning [1] and has been demonstrated in a variety of species including rat [2, 3], rabbit [4], and swine [5]. Our laboratory [6] and others [7–9] have recently demonstrated that human myocardium can also be preconditioned. The observation that preconditioning of the human myocardium can be accomplished therapeutically suggests a potential clinical role for the elective induction of this endogenous protective strategy. Current enthusiasm surrounding warm cardioplegic myocardial revascularization has increased the potential for inadvertent normothermic cardiac ischemic injury. Similarly, retrograde cardioplegia delivery has repeatedly been criticized as providing inadequate hypothermic protection of the right atrium and ventricle. Examination of strategies to protect human myocardium against normothermic ischemia plus reperfusion appears uniquely current and relevant.
Cave and Hearse [10] demonstrated that ischemic preconditioning protects contractile function against hypothermic (20°C) ischemic injury. Engelman and colleagues [11] also reported that a transient period of hypoxia in rat heart improves functional recovery after 4 or 6 hours of cold cardioplegic arrest. Electively induced cardioadaptation of human myocardium with transient ischemia against a subsequent ischemic injury has been incompletely explored, however. Hypothermic ischemia at 4°C is mandatory during donor heart procurement and transportation for heart transplantation. Further, Keon and associates [12] previously reported that combined hypothermic and ischemic injury at 4°C induces unavoidable contractile dysfunction in human myocardium. The purposes of this study, therefore, were to determine whether ischemic preconditioning protects contractile function of human myocardium against normothermic ischemia-reperfusion injury and whether ischemic preconditioning promotes myocellular viability in human myocardium subjected to a hypothermic or normothermic ischemia-reperfusion injury.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Each appendage was placed in oxygenated modified Tyrode's solution at 4°C. Two trabeculae (diameter, <1.0 mm, and length, 4 to 7 mm) were obtained from each appendage and suspended vertically in an organ bath between two clips. The bottom clip was fixed, and the top clip was attached to a force transducer. Each organ bath contained 30 mL of modified Tyrode's solution, which was bubbled (40 mL/min) with a 92.5% O2 and 7.5% CO2 gas mixture providing normoxic superfusion. Solution gas tensions and pH were maintained at an O2 tension of greater than 360 mm Hg, a CO2 tension of 38 to 42 mm Hg, and a pH of 7.35 to 7.45, which were monitored with an automated blood gas analyzer (ABL Instruments, Copenhagen, Denmark). Temperature in the organ baths was maintained at 37.5°C. During the simulated ischemic period, the gas mixture was switched to 92.5% N2 and 7.5% CO2, which produced an O2 tension of less than 50 mm Hg, and the organ baths were covered to prevent atmospheric gas exchange. The Tyrode's buffer was replaced at 20-minute intervals throughout experimentation except during the period of simulated ischemia.
A 30-minute stabilization period was allowed for each trabecula after mounting. The optimal length–tension (preload) for human atrial trabeculae in our laboratory has been previously identified as a resting force of 1 g. Platinum electrodes (Radnoti Glass, Inc, Monrovia, CA) provided field stimulation at a frequency of 1 Hz. The platinum electrodes were positioned on either side of each trabecula and were driven with a SD9 stimulator (Grass, Warwick, RI) with 5-ms pulses at a voltage of 10% higher than threshold. Isometric contractile responses were detected by Grass FT03 force-displacement transducers and recorded with a computerized preamplifier/digitizer (MacLab 8; AD Instruments, Milford, MA) and a Macintosh computer (Apple Computer, Cupertino, CA). The indices of contractile function assessed were developed force (DF) and resting force, both measured in grams. Before the study, standards were established to discard trabeculae that failed to generate at least 0.5 g of DF during the initial equilibration period, but no trabeculae were excluded.
The modified Tyrode's solution was prepared daily with deionized distilled water and consisted of the following in millimoles per liter: D-glucose, 5.0; CaCl2, 2.0; NaCl, 118.0; KCl, 4.0; MgSO4•7H2O, 1.2; NaHCO3, 25.0; and NaH2PO4, 1.2. All reagents were from Sigma Chemical Company. In the substrate-free Tyrode's solution, choline chloride (7 mmol/L) was added to maintain constant osmolarity.
Experimental Design
WARM ISCHEMIA.
Trabeculae were subjected to a 60-minute equilibration period to allow stabilization of DF, and subsequently all experiments were conducted for 180 minutes. Trabeculae (Warm I/R) (n = 5) were subjected to 15 minutes of normoxic perfusion, 45 minutes of simulated ischemia (hypoxic, substrate-free buffer with pacing at 3 Hz), and 120 minutes of reperfusion (normoxic buffer with glucose and pacing at 1 Hz). Preconditioned trabeculae (Warm IPC) (n = 5) received 5 minutes of normothermic simulated ischemia followed by 10 minutes of normoxic perfusion before 45 minutes of simulated ischemia and 120 minutes of reperfusion. Control trabeculae (n = 3) were perfused with normoxic Tyrode's buffer for 180 minutes. At the end of reperfusion, all trabeculae were removed from the organ baths, weighed, measured, and rapidly frozen in liquid nitrogen for tissue creatine kinase (CK) activity. Samples were stored at -70°C, and CK assay was performed within 2 weeks.
HYPOTHERMIC ISCHEMIA.
Trabeculae were subjected to a 60-minute equilibration period to allow stabilization of DF, and subsequently all experiments were conducted for 315 minutes. Trabeculae (Cold I/R) (n = 5) were subjected to 15 minutes of normoxic perfusion and then arrested with St. Thomas' solution and subjected to 240 minutes of hypothermic ischemia (4°C) by cooling the organ baths, which contained Tyrode's solution devoid of glucose. The trabeculae were then rewarmed to 37°C and reperfused for 60 minutes. Preconditioned trabeculae (Cold IPC) (n = 5) received 5 minutes of normothermic simulated ischemia followed by 10 minutes of normoxic perfusion before 240 minutes of hypothermic ischemia (4°C) and 60 minutes of warm reperfusion. Control trabeculae (n = 3) were perfused with normoxic Tyrode's buffer for 315 minutes. At the end of reperfusion, all trabeculae were removed from the organ baths, weighed, measured, and rapidly frozen in liquid nitrogen for tissue CK activity. Samples were stored at -70°C, and CK assay was performed within 2 weeks.
Trabecular Tissue Creatine Kinase Activity
End-reperfusion tissue CK activity was determined as previously described in our laboratory [13]. In brief, trabeculae were added to 10 volumes of cold isotonic extraction buffer consisting of the following in millimoles per liter: imidazole acetate, 50; Mg2+ acetate, 10; KH2PO4, 4; EDTA (ethylenediaminetetraacetic acid), 2; N-acetylcysteine, 0.05; and sulfur in 0.8% ethanol, 0.012; pH 7.6. Samples were homogenized with a vertishear tissue homogenizer (parallel blades 0.5 cm apart) at half the maximal speed for 20 seconds (ten equally spaced bursts) followed by centrifugation at 2,000 g for 5 minutes and 20,000 g for 10 minutes. The final supernatant was diluted to less than 0.25 absorbance units per minute. The assay was performed with Sigma diagnostic kit No. 47-UV on an automated spectrophotometer (Centrifichem 500 discrete autoanalyzer; Union Carbide, Palo Alto, CA) in cuvettes maintained at 30°C. Samples and reagents were maintained at 4°C prior to assay. Results are presented as units of CK activity per gram of wet weight of tissue.
Statistical Analysis
All data are presented as the mean ± the standard error of the mean. All values were compared using repeated-measures analysis of variance with application of a post hoc Bonferroni/Dunn test. A p value of less than 0.05 was accepted as representing a difference between groups.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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. As baseline DF was similar among all experimental trabeculae, DF data are presented as a percentage of baseline DF, which was recorded at the end of equilibration.
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. The preconditioning stimulus of simulated ischemia provoked a decrease in DF to 46% ± 5% of baseline after 5 minutes (p < 0.05 versus Warm I/R). The DF in the Warm IPC trabeculae recovered to 90% ± 4% of baseline before the 45-minute normothermic simulated ischemic injury. During the 45 minutes of normothermic simulated ischemic injury, both the Warm IPC and Warm I/R trabeculae exhibited a rapid fall in baseline DF, which did not differ between the two groups. At the end of reperfusion, however, the Warm IPC trabeculae recovered a greater amount of baseline DF versus Warm I/R trabeculae (51% ± 5% versus 24% ± 3%; p < 0.05). Control trabeculae maintained 86% ± 5% of baseline DF at 180 minutes of normoxic perfusion.
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. Warm IPC trabeculae contained 1,183 ± 204 U/g wet myocardium, whereas Warm I/R trabeculae had 403 ± 32 U/g wet myocardium at the end of reperfusion (p < 0.05). Tissue CK activity in freshly isolated trabeculae not subjected to ischemia plus reperfusion was 1,973 ± 394 U/g wet myocardium (n = 3).
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. The initial simulated ischemic preconditioning stimulus provoked a similar fall in DF to 47% ± 4% after 5 minutes in the Cold IPC trabeculae (p < 0.05) versus Cold I/R trabeculae, which is similar to the finding in the Warm IPC group. After 10 minutes of perfusion, DF recovered to 91% ± 3% in the Cold IPC trabeculae. After 4 hours of hypothermic (4°C) ischemic injury, both Cold IPC and Cold I/R trabeculae were similarly protected with recovery of DF to 58% ± 6% in the Cold IPC trabeculae and 52% ± 4% in the Cold I/R group (p > 0.05). By 15 minutes of warm reperfusion, both groups began to show a marked fall in percent DF, which reached a nadir of 15% ± 5% in Cold IPC trabeculae and 19% ± 5% in Cold I/R trabeculae. At no time during the warm reperfusion period was the percent recovery of the Cold IPC trabeculae greater than that of the Cold I/R trabeculae, however. Control trabeculae retained 77% ± 6% of baseline DF at 315 minutes of normoxic perfusion.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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The protection afforded by ischemic preconditioning in human myocardium subjected to normothermic ischemic injury is depicted in Figure 1
. The initial fall in DF induced by the preconditioning stimulus resolved by the initiation of normothermic ischemic injury. Although ischemic preconditioning did not protect contractile function during the 45 minutes of simulated ischemia, the preconditioning stimulus did protect function on reperfusion. Our observations of protection of contractile function by preceding simulated ischemia are in agreement with the results of Walker and colleagues [7], who found similar protection in an in vitro model of human atrial trabeculae preconditioning. The chosen injury of 45 minutes of simulated ischemia reliably allows a consistent recovery of DF in I/R trabeculae and has been used by others [7] for the study of human preconditioning. We believe that the enhanced contractile function in the Warm IPC trabeculae could be, in part, a result of increased myocellular viability as reflected by augmented tissue CK activity (see Fig 2) at end-reperfusion. This association between myocellular viability and tissue CK activity is arguably indirect, and tissue viability is dependent on other factors besides tissue CK activity.
Protection of contractile function in human myocardium against a prolonged (4-hour) hypothermic (4°C) and subsequent warm reperfusion injury was not further enhanced by a preceding 5-minute simulated ischemic stimulus (see Fig 3). As in the warm ischemia group, the 5-minute simulated ischemic preconditioning stimulus provoked a fall in DF that had resolved on initiation of 4 hours of hypothermic ischemic injury. The contractile function of both Cold IPC and Cold I/R trabeculae was depressed on initial reperfusion (time 0 in Fig 3). However, during early warm reperfusion, both Cold IPC and Cold I/R trabeculae manifested an initial increase in percent DF (time 5 minutes in Fig 3) relative to the initiation of warm reperfusion, followed by a marked loss of percent DF (from time 5 minutes to time 20 minutes). The two groups of trabeculae (Cold IPC and Cold I/R) then recovered their percent DF, but in both, the percentage was depressed relative to baseline after 60 minutes of normothermic reperfusion. Importantly, the ischemic preconditioning stimulus failed to protect the contractile function or tissue CK levels at any point during warm reperfusion after hypothermic ischemic injury.
Hypothermic ischemic injury occurs universally as a component of heart procurement and transportation for cardiac transplantation, and resultant contractile dysfunction has been reported previously in human myocardium [12]. The results of the present study concur with those of Keon and colleagues [12], who evaluated the contractile response of human atrium to hypothermic ischemic injury. This group observed a similar loss of contractile function after 4 hours of 4°C ischemic injury in human atrial trabeculae. The present study builds on their findings by reporting the effects of a preconditioning stimulus on recovery of contractile function in human trabeculae exposed to hypothermic ischemia.
Our observations suggest that normothermic preconditioning with simulated ischemia does not modify the degree of contractile dysfunction after hypothermic ischemia. We chose the time course of 4 hours of hypothermic ischemia because this point approaches the maximally permissible cold ischemic period for donor heart storage and transport. In a subsequent study by Deslauriers and associates [14], the contractile dysfunction after hypothermic (4°C) ischemic injury was associated with preserved tissue adenosine triphosphate levels. Our observations combined with those previously reported suggest that contractile dysfunction occurs in human myocardium after 4 hours of hypothermic ischemic injury and that metabolic and mechanical function may be dissociated.
Investigations regarding protection elicited by ischemic preconditioning against hypothermic ischemic injury are limited. Cave and Hearse [10] explored protective ischemic preconditioning (5 minutes' ischemic stimulus) against a hypothermic (20°C) and normothermic ischemic injury in an isolated working rat heart model. They found protection against both injuries. The protection against hypothermic ischemia spanned a range of 115 to 160 minutes of ischemic time. Of note, the hearts in their study were not arrested with cardioplegic solution prior to hypothermic ischemia. In a similar model of an isolated working rat heart preparation, Engelman and colleagues [15] also demonstrated protection against 4 or 6 hours of hypothermic (4°C) storage. In their study, hearts were arrested with St. Thomas' cardioplegic solution and then cooled to 4°C for 4 or 6 hours. They observed that a 10-minute period of hypoxic preconditioning prior to hypothermic ischemia protected recovery of aortic flow (mechanical function), was associated with lessened intracellular sodium and calcium, and induced heat shock protein 70 and catalase messenger ribonucleic acid.
There are important differences in study design between the present study and prior investigations of preconditioning against hypothermic ischemia. The model we chose was isolated human atrial trabeculae, a model different from an intact isolated heart preparation. This trabecular model has proved useful for the study of preconditioning in human ventricular [6] and atrial [7, 8] myocardium. The end point examined in the present study, however, was recovery of DF. Recovery of aortic flow, which was reported in the previous investigations, necessitates a more complex interplay of coronary circulation and myocardial contractile state. It is conceivable that DF as the outcome variable may not discriminate differences that provide protection in whole-heart preparations.
The use of human atrial tissue and our choice of simulated ischemia as an injury also deserve comment. Atrial tissue does differ from ventricular tissue in metabolic [16] and physiologic [17, 18] function. It is possible that our results are specific to human atrial tissue. However, previous work from our laboratory [6] indicated that ischemic preconditioning prior to a normothermic hypoxic injury in diseased, explanted human ventricular trabeculae yields qualitatively similar protection to that seen in the present study. Further, human right atrial trabeculae are an attractive model for studying mechanisms of preconditioning in human myocardium because of their availability and because they represent a stable preparation for study of contractile function. The simulated ischemia injury models three components of ischemia-deprivation of oxygen, deprivation of substrate, and accumulation of metabolic waste. Clearly, however, we cannot directly study ischemia in this model, as the isolated trabeculae rely on diffusion of oxygen and substrates rather than perfusion through intact vessels. During simulated ischemia, the diffusion of oxygen and nutrients is limited. This differs from the lack of perfusion that occurs with occlusion of a coronary artery or aortic cross-clamping in situ.
The tissue CK activity data also offer insight into the differential response of preconditioning to hypothermic or normothermic ischemic injury. Although this variable only indirectly assesses the viability of myocytes, it has been previously reported that tissue CK is also sensitive to oxidant stress [13, 19]. Of interest, tissue CK activity in the Cold I/R trabeculae was lower than in the Warm I/R trabeculae, but the recovery of DF after 60 minutes of warm reperfusion in Cold I/R trabeculae was greater than in the Warm I/R trabeculae (see Figs 1, 3). Although the tissue CK data do not clearly discriminate the reversibility of hypothermic or normothermic ischemic injury, the augmented levels of tissue CK in the Warm IPC trabeculae compared with the Warm I/R trabeculae implicate either a metabolic benefit or enhanced viability conferred by ischemic preconditioning. A metabolic basis of protection for ischemic preconditioning has also been suggested by others [20]. The intriguing observation that tissue CK activity does not correlate with contractile recovery after hypothermic ischemia suggests a differential metabolic response to normothermic and hypothermic ischemic injuries.
The potent endogenous protection of ischemic preconditioning may have clinical relevance in the setting of cardiac surgery and heart transplantation. This relevance may be enhanced by virtue of current enthusiasm for normothermic cardioplegic strategies. The observation that a differential effect of ischemic preconditioning exists against normothermic or hypothermic ischemic injury in human myocardium is important. Caution must be exercised in extrapolating these in vitro results to clinical applications. The modeling conditions used in this study differ from several factors present in the human heart in situ. Clearly, further investigation is warranted to determine the mechanisms underlying this differential response. Whether the ischemic preconditioning stimulus can be optimized to deliver protection against a warm reperfusion injury after a hypothermic insult remains to be determined.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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References |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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1-adrenergic mechanism. Circ Res 1993;73:656–70.Related Article
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