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Ann Thorac Surg 1996;62:543-548
© 1996 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery, Department of Surgery, The University of Texas Medical Branch, Galveston, Texas
Accepted for publication April 4, 1996.
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. To test this hypothesis, we subjected isolated, perfused rabbit hearts to either one 5-minute transient ischemic period or three 5-minute transient ischemic periods followed by a 40-minute period of warm ischemia and 30 minutes of reperfusion. Control hearts (no pretreatment with transient ischemia) were examined simultaneously. Left ventricular developed pressure was measured with an intraventricular balloon.
Results. Postischemic recoveries (expressed as percent of preischemic values) of left ventricular developed pressure for the group with one ischemic period and the group with three ischemic periods were 43% ±- 5% (n = 5) and 38% ±- 6% (n = 6), respectively. These values were not significantly different from control values.
Conclusions. Neither one nor three periods of transient ischemia protect the isolated, perfused rabbit heart from postischemic contractile dysfunction. Therefore, the rabbit heart may not have the capacity to be ischemically preconditioned against postischemic contractile dysfunction.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Brief, transient ischemia (TI) results in metabolic changes that can render heart muscle more tolerant to a subsequent prolonged ischemic episode. This phenomenon, known as ischemic preconditioning, is one of the most potent experimental interventions that can limit myocardial necrosis. Several studies from many different laboratories have clearly shown that dog [1], pig [2, 3], rabbit [4], and rat [5] hearts can be preconditioned against necrosis. The efficacy of ischemic preconditioning in experimental models has led to hope that the protective mechanisms could be used in the clinical setting. However, the potential usefulness of ischemic preconditioning to protect the heart against postischemic contractile dysfunction has not clearly been determined. Only in the isolated, perfused rat heart is there convincing evidence that ischemic preconditioning protects against postischemic contractile dysfunction [6–9]. Our previous studies [6, 10] have shown that the ischemically preconditioned rat heart (compared with the nonconditioned heart) is characterized by enhanced left ventricular developed pressure (LVDP) and lower left ventricular end-diastolic pressure (LVEDP) upon reperfusion after a prolonged period of normothermic ischemia. The magnitude of protection was substantial, with preconditioned hearts recovering postischemic developed pressure approximately twice that of nonconditioned hearts [6, 10]. These preconditioned rat hearts may tolerate prolonged ischemia because the drop in myocardial pH during the ischemic period is attenuated [6, 8, 10, 11]. Whether cardioprotection against postischemic contractile dysfunction is unique to the rat heart or to the isolated perfused heart model has yet to be established.
There are contradictory reports regarding the ability of TI to precondition the isolated rabbit heart against postischemic contractile dysfunction. Although two studies [12, 13] showed that TI did not improve postischemic LVDP or LVEDP, another study [14] showed that TI improved both postischemic LVDP and LVEDP of the isolated rabbit heart. Partial protection was demonstrated by Lasley and Mentzer [15], who reported that TI did not improve postischemic LVDP, but did improve LVEDP in the initial reperfusion period.
The mixed results of the rabbit studies may indicate that although the rabbit heart can be preconditioned against postischemic contractile dysfunction, the threshold of the stimulus may be higher in the rabbit than the rat heart. If true, this would apparently contradict the conclusion of Liu and Downey [5] that the threshold for preconditioning against necrosis is lower in the rabbit heart than the rat heart. These investigators came to this conclusion because they found that although a single cycle of TI would protect against necrosis in the rabbit heart, three cycles of TI were required to precondition the rat heart against necrosis [5]. However, because the cardioprotective mechanisms against necrosis and contractile dysfunction may be different, the relative susceptibilities of rat and rabbit hearts to be preconditioned may differ with respect to the two end-points. In those studies using the isolated rabbit heart looking at the effect of ischemic preconditioning on contractile dysfunction, the preconditioning stimulus used was a single cycle of TI [12–15].
The purpose of the present study was to determine if three cycles of TI can protect the isolated, perfused rabbit heart against postischemic contractile dysfunction better than a single cycle of TI. In addition, we wanted to determine if, as with rat heart, pretreatment with TI can attenuate the accumulation of H+ during prolonged ischemia.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Isolated, Perfused Rabbit Heart
Male New Zealand White rabbits weighing between 2 and 3 kg were first sedated with an intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg). The rabbits were anesthetized with an intravenous injection of sodium pentobarbital (15 to 20 mg/kg). The rabbits were then given an intravenous injection of heparin (100 units/kg). The hearts were quickly exposed, excised, and placed in ice-cold saline solution. The aorta was quickly secured to a polyethylene cannula, and the heart was perfused with Krebs-Henseleit buffer using a nonrecirculating Langendorff preparation. The millimolar concentrations of constituents of the Krebs-Henseleit buffer were as follows: KCl, 4.7; CaCl2, 1.2; MgCl2, 1.25; KH2PO4, 1.25; NaHCO3, 25; NaCl 118; and glucose, 10. The buffer was filtered through 0.45-µm cellulose filters immediately after preparation to remove any particulate matter. The temperature of the buffer was kept constant at 38°C in a water-jacketed column and gassed continuously with a 95% oxygen, 5% carbon dioxide mixture. The perfusion pressure was kept constant at 100 cm H2O.
A small incision was made in the center of the left atrium. An apical stab was made through the left ventricle with a 14-gauge needle. A latex balloon was inserted into the left ventricle through the left atrium and tied securely into place. The balloon was filled with water to give an end-diastolic pressure between 5 and 10 mm Hg. The balloon volume remained constant throughout the experiment. The balloon was connected to a catheter-tipped pressure transducer (Camino Laboratories, San Diego, CA) via fluid-filled polyethylene tubing. The transducer was interfaced to a Gateway 2000 486 4DX2-66V computer. Hemodynamic data were collected and analyzed using Cardiology Research Data Acquisition [16] version 1.05 (Symbolic Logic, Dallas, TX). Hemodynamic data for each time-point analyzed were averaged for five consecutive beats. Ventricular function was assessed by LVEDP and LVDP. Left ventricular developed pressure is defined as peak systolic minus end-diastolic pressure. During the initial equilibration period, a 21-gauge pH microelectrode (Kent Scientific Corp, Litchfield, CT) was carefully inserted into the mid-myocardium of the left ventricular wall to monitor interstitial pH, which is directly related to intracellular pH [17]. Coronary flow rates were determined by collecting the coronary effluent in a graduated cylinder. Ischemia was induced by closing a stopcock in the perfusion line immediately above the aortic cannula. During ischemia, the hearts were immersed in Krebs-Henseleit buffer contained in water-jacketed chamber maintained at 37°C.
Experimental Protocols
Studies were performed to determine the effects of a single cycle and triple cycles of TI episodes on postischemic recovery of hemodynamic functions. Single and triple TI cycles were studied in two separate studies. In both studies, control (CN) and TI hearts were examined simultaneously. Six hearts were assigned to each group. Figure 1
summarizes the experimental protocols for both studies.
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TRIPLE-CYCLE TRANSIENT ISCHEMIA STUDY.
Control (CN-3) hearts were perfused for 50 minutes then made ischemic for 40 minutes. Hearts assigned to the TI group (TI x 3) were perfused for 15 minutes then treated with three periods of 5 minutes of ischemia interrupted by 5 minutes of reperfusion. After the third 5-minute ischemic period, the hearts were perfused for 10 minutes followed by 40 minutes of ischemia. All hearts were reperfused for 30 minutes after the 40 minute ischemic period. Baseline values were taken after 15 minutes of perfusion.
Statistical Analysis
Data are reported as means ±- standard error of the mean. Unpaired Student's t test was used to test for differences between groups. A value of p less than 0.05 was considered significant.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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). Figure 2 shows the LVDP for each group throughout the experimental protocol. Single or triple TI did not significantly improve postischemic (after the 40-minute ischemic period) LVDP. There was a trend toward improvement after triple TI (see Fig 2, right panel), but the differences were not statistically significant at any of the three time points indicated during reperfusion.
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shows the postischemic LVEDP for each group during the 30-minute reperfusion period following 40 minutes of ischemia. Postischemic LVEDP was not significantly affected by pretreatment with TI. A single cycle of TI tended to result in lower LVEDP compared with the CN values (see Fig 3, left panel). Moreover, the CN-1 hearts tended to have a greater degree of reperfusion contracture compared with the other groups (see Fig 3). However, the differences were not statistically significant.
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). To determine if TI pretreatment significantly affected the degree of reperfusion-induced contracture, the differences between LVEDP at 10 minutes of reperfusion and the left ventricular pressure at the end of 40 minutes of ischemia were analyzed (Fig 4). A single cycle of TI significantly reduced the degree of reperfusion-induced contracture. Although triple-cycle TI did not affect reperfusion-induced contracture compared with the CN-3 group, the values for the CN-3 and TI x 3 groups were significantly lower than that of the CN-1 group (see Fig 4).
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shows the myocardial pH for the hearts throughout the experimental protocols in the two studies. In all hearts, ischemia resulted in a decrease in pH, which was readily reversible by reperfusion. Pretreatment with a single cycle of TI did not affect the rate or extent of acidosis during the 40-minute ischemic period (see Fig 5, left panel). For the triple-cycle study, the pH tended to be higher in the TI x 3 hearts compared with CN-3 hearts between 15 and 30 minutes of ischemia (see Fig 5, right panel). However, the differences were not statistically significant.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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). Although the differences between TI-treated and CN hearts with respect to postischemic end-diastolic pressure were not statistically different, the absolute change in end-diastolic pressure after reperfusion was significantly lower in the TI-treated hearts (see Fig 5, single-cycle study), indicating better diastolic function at least during the initial phase of reperfusion. Another important finding of this study is that postischemic hemodynamic functions of hearts pretreated with three cycles of TI were not significantly improved compared with control values. This implies that not only does enhanced stimulus with several periods of TI not provide additional protection, but any protection afforded by a single period may be lost when the rabbit heart is subjected to additional TI cycles.
Similar to the present study, Lasley and Mentzer [15] did not find improvement in postischemic developed pressure with TI pretreatment, but did find a significant improvement in postischemic end-diastolic function in single-cycle TI-treated rabbit hearts during initial reperfusion (
10 minutes). However, no significant protection was seen after reperfusion [15] for greater than 10 minutes. We did not find a statistically significant difference between the postischemic end-diastolic pressures of CN and TI-treated hearts at any time point measured, including the 10-minute time point. This discrepancy may be explained by the differences in the length of the prolonged ischemic period. In their study [15], the prolonged ischemic time was 60 minutes, which may have been sufficient time for all of the hearts to be in ischemic contracture at the time of reperfusion. In our study, there was a variable degree of contracture at the time of reperfusion. This large variation in ventricular pressure at the time of reperfusion may explain why a trend toward protection against diastolic dysfunction throughout reperfusion was seen but was not statistically significant (see Fig 3). Qualitatively, the results of our study and that of Lasley and Mentzer [15] are similar. Both studies suggest that although TI may be able to improve postischemic diastolic dysfunction upon early reperfusion, the rabbit heart cannot be ischemically preconditioned against postischemic systolic dysfunction.
However, a review of the literature reveals that the ability of TI to precondition the isolated rabbit heart against postischemic contractile dysfunction is controversial. Sandhu and colleagues [12] and Quantz and co-workers [13] failed to find any protection of a single cycle of TI against either systolic or diastolic function. Conversely, Hendrikx and associates [14] reported that both postischemic developed pressure and end-diastolic pressure were improved if the hearts were pretreated with a single cycle of TI. Similar to this latter study, Omar and associates [18, 19] reported improved postischemic developed pressure in hearts pretreated with a single cycle of TI.
Because the conflicting results cannot be explained by species differences, variations in experimental protocols are likely to explain the different results. The different results cannot be explained by different preconditioning stimuli, because the transient ischemic time was 5 minutes in all of the studies. There were differences in the length of the prolonged ischemic time, which varied from 30 to 60 minutes in these studies [12–15, 18, 19]. However, this is unlikely to be the cause of the contradictory results because Lasley and Mentzer [15] and Omar and associates [18, 19] both used 60 minutes of prolonged ischemia and still reported different results. The other experimental protocol that varied among the different studies was the initial stabilization period on the perfusion apparatus before any interruption of coronary flow. Analyses of the various studies did indicate an inverse relationship between the time the hearts were on the perfusion apparatus before the prolonged ischemic period and the ability of TI to precondition against postischemic dysfunction as measured by LVDP and LVEDP (Table 2). In the studies [14, 18, 19] where this time was only 15 minutes, a preconditioning effect was clearly seen (see Table 2). In those studies [13, this study triple-cycle TI] where the time to prolonged ischemia was 50 minutes or greater, no preconditioning effect was seen. In those studies [12, 15, this study single-cycle TI] where the time to prolonged ischemia was intermediate (20 to 35 minutes), no protection was observed with respect to LVDP, but protection against early postischemic diastolic dysfunction was observed in two of the three studies [15, this study], implying a possible small benefit from ischemic preconditioning. Collectively, the data suggest that conflicting reports on the ability of TI to precondition the isolated, perfused rabbit heart against postischemic contractile dysfunction may in part be due to the use of different stabilization times after cannulation of the aorta. However, this is certainly speculative and further studies are necessary to verify this possibility.
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). The inability of TI to attenuate acidosis during prolonged ischemia may explain the lack of protection against postischemic contractile dysfunction in the rabbit heart. Other differences between rat and rabbit hearts with respect to ischemic preconditioning have been reported. Although rat and rabbit hearts can both be ischemically preconditioned against infarction, adenosine plays a significant role in this protection in the rabbit [4] but not the rat heart [6, 7, 20]. In addition, whereas we have shown that the rat heart can be preconditioned against postischemic contractile dysfunction with transient pretreatment with dobutamine [21] and norepinephrine [22], the rabbit heart cannot (unpublished data). It is not clear why rabbit and rats are so different with respect to ischemic preconditioning. The apparent inability to precondition the rabbit heart against postischemic contractile dysfunction does not support the idea that ischemic preconditioning would benefit myocardial recovery in the clinical setting. However, understanding these species differences may be helpful in elucidating the mechanism of ischemic preconditioning and developing better interventions to improve postischemic contractile function.
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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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