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Ann Thorac Surg 2000;70:609-613
© 2000 The Society of Thoracic Surgeons

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Original articles: cardiovascular

Amrinone preconditioning in the isolated perfused rabbit heart

^a Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
^b Division of Cardiothoracic Surgery, University Hospital and Medical Center, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York, USA

Address reprint requests to Dr Saltman, Department of Surgery, State University of New York at Stony Brook, Health Sciences Center T19–080, Stony Brook, NY 11794–8191
e-mail: saltman{at}surg.som.sunysb.edu

	Abstract

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Background. Ischemic preconditioning (IPC) reduces infarct size in experimental preparations. IPC, however, is not without detrimental effects. We studied amrinone as a possible alternative to IPC.

Methods. Isolated perfused rabbit hearts were given a 5-minute infusion of 10 µmol/L amrinone followed by a 5-minute washout (n = 6). The anterior descending artery was then occluded for 1 hour and reperfused for 1 hour. Six hearts underwent IPC, with two episodes of 5-minute global ischemia followed by 5-minute reperfusion before LAD occlusion; eight control hearts received no preconditioning. Left ventricular pressure and ischemic zone epicardial monophasic action potentials were continuously monitored.

Results. IPC but not amrinone reduced peak pressure before anterior descending artery occlusion. Peak pressure fell significantly during ischemia and reperfusion in all hearts. End diastolic pressure rose significantly during reperfusion in control and IPC hearts but not in amrinone hearts. Action potentials shortened during ischemia in all hearts. They returned to preocclusion values in control hearts but lasted beyond preocclusion values in IPC and amrinone hearts. Both the incidences of ventricular fibrillation and infarct size were significantly reduced in amrinone hearts but not in IPC hearts.

Conclusions. Amrinone is not only a useful inotropic agent but is also a superior preconditioning agent when compared to IPC.

	Introduction

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Transient episodes of stress have been found to elicit a protective reflex against prolonged ischemia in many experimental animal species [1–9]. This reflex has been termed preconditioning [1] and was described by the use of transient global ischemia to reduce the infarct size resulting from subsequent ischemic insults. Since its original description, many other stimuli in addition to transient ischemia have been successfully used to trigger the preconditioning response, including rapid pacing [2], cardiopulmonary bypass [3], and drugs such as -adrenergic agonists [4], ß-adrenergic agonists [5, 6], activators of protein kinase C [7], adenosine [8], and adenosine triphosphate-sensitive potassium channel openers [9]. Despite these multiple stimuli, the precise subcellular mechanism underlying preconditioning remains undefined.

Recent studies concerning the effects of preconditioning on intracellular messengers such as calcium [10] and 3',5'-cyclic adenosine monophosphate (cAMP) [2, 11–14] have revealed that the levels of these compounds are altered in preconditioned hearts. This is not surprising, as many of the agents shown to elicit the preconditioning response are thought to function through receptors coupled to G proteins and thereby modulate the activity of adenylate cyclase. It remains unknown, however, whether specific manipulation of intracellular levels of cAMP would prove effective at triggering the preconditioning response.

In the clinical sphere, adrenergic agonists are commonly employed to help support the failing heart, particularly after coronary artery bypass surgery. These drugs presumably increase intracellular levels of cAMP and initiate a cascade of events that results in increased intracellular calcium influx during the action potential, with resultant increased inotropy. One such drug is amrinone, one of the phosphodiesterase inhibitors. Amrinone, in distinction to other positive inotropic agents, does not increase myocardial oxygen consumption [15–17], nor does it result in a tachycardic response in the whole organism [16]. These characteristics have made it an attractive agent for use in the clinic, and may make it desirable for use as a preconditioning agent. We therefore conducted this study to quantify amrinone’s electrophysiologic and functional preconditioning properties and to compare them to those of ischemic preconditioning.

	Material and methods

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Experimental setup
Twenty-four adult male New Zealand albino rabbits were anesthetized with sodium pentobarbital (30 mg/kg) and heparin (1000 IU) via ear vein. Their hearts were rapidly excised through a transverse thoracotomy and placed in 4°C Krebs-Henseleit buffer (in mmol/L: Na⁺ 135, K⁺ 4.7, Ca²⁺ 1.7, PO₄^- 1.1, Mg 1.2, HCO₃^- 25, glucose 11.5, pyruvate 4.9, and fumarate 5.4) gassed with 95% O₂/5% CO₂. The aorta was cannulated and the heart suspended on a Langendorff perfusion apparatus. The coronary arteries were perfused with oxygenated 37°C Krebs solution at 75 mm Hg pressure.

Both atria were removed and the heart paced at 150 beats per minute (Model 5880A, Medtronic, Minneapolis, MN). A balloon-tipped pressure gauge (Millar Instruments Inc, Houston, TX) was placed in the left ventricle. An epicardial monophasic action potential probe (EP Technologies model 200, Sunnyvale, CA) was placed lightly against the left ventricle in the area supplied by the anterior descending coronary artery. The anterior descending artery and the first obtuse marginal artery (referred to as LAD) were then surrounded close to their bifurcation with a 3-0 silk suture and a Rummel tourniquet. The entire assembly was then immersed in a heated jacket (Radnoti Glass Technologies Inc, Monrovia, CA) filled by coronary effluent.

Measurements
After a 30-minute equilibration period, baseline data were obtained for 7.5 seconds. These data sets consisted of left ventricular epicardial monophasic action potentials, left ventricular endocavitary pressure, and coronary flow, which was measured directly in a graduated cylinder. The measurements were repeated after each intervention and at 15-minute intervals throughout the experiment.

Experimental protocol
Eight control hearts received no preconditioning, and underwent 1 hour of LAD occlusion followed by 1 hour of reperfusion. Seven ischemically preconditioned hearts (IPC) underwent two cycles of global preconditioning consisting of 5 minutes of aortic inflow occlusion followed by 5 minutes of reperfusion with oxygenated Krebs solution. Five amrinone-preconditioned hearts underwent a single infusion of 10 µmol/L amrinone for 5 minutes followed by a 5-minute washout period with oxygenated Krebs solution. After preconditioning was complete, the LAD was occluded for 1 hour and then reperfused for 1 hour. If the heart fibrillated at any time during the experiment, it was defibrillated with the strike of a fingernail.

Upon completion of the reperfusion period, the LAD was ligated and 2 mL of phthalocyanine blue (Engelhard Corp, Louisville, KY) was infused into the aortic root. The area at risk did not stain with the dye. The right ventricle was dissected away and the heart was horizontally sectioned into slices 1 to 2 mm thick. The slices were incubated in triphenyl tetrazolium chloride (Sigma Chemical Co, St. Louis, MO) for 15 minutes at 37°C. Viable tissue stained pink. Infarct area was determined as a percentage of the area at risk using the formula %Infarct = Area of Infarct (white tissue)/Area at Risk (white tissue + pink tissue) by two-dimensional planimetry using a scanning tablet (Summadraw, Summagraphics, Seymour, CT) and commercially available software (SigmaScan, Jandel Scientific, San Rafael, CA).

Data analysis
Recordings taken from the pressure gauge and the action potential probe were digitized by an 8-bit analog-to-digital converter (Model 2901, Data Translation Inc, Marlborough, MA) and recorded to the hard disk of an IBM PC-compatible personal computer (Model 450, Dell Corp., Austin, TX). A digital spreadsheet (Microsoft Excel version 5.0, Microsoft Corp, Redmond, WA) using custom-designed macro programs determined the systolic and diastolic pressure and action potential duration of each beat. Pressures and action potential durations are reported as mean values taken over the 7.5-second recording period.

Comparisons among groups were made with multiple ANOVA (Systat version 5.02, Systat, Inc, Evanston, IL). Significance was determined at the p less than 0.05 level using the Tukey posthoc test. Comparisons of time points within each group were made similarly with multiple ANOVA after applying the Bonferroni correction. Categorical data were compared with ² analysis. Nonparametric comparisons were performed with the Kruskal-Wallis one-way ANOVA. All data are presented as mean ± standard error (SEM).

	Results

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Electrophysiologic effects of amrinone preconditioning
Changes in the action potential configuration were common to all experimental groups (a representative example is shown in Figure 1). First, neither ischemic preconditioning nor amrinone preconditioning alone significantly altered the action potential duration at 50% repolarization (IPC: 75 ± 3 to 82 ± 6 ms; amrinone: 74 ± 5 to 83 ± 7 ms). Second, during regional ischemia, the action potential duration shortened significantly (p < 0.05) in all groups, without any differences among them. Third, during reperfusion the action potential duration returned to baseline, again without significant differences among groups (IPC: 82 ± 6 to 92 ± 4 ms; amrinone: 83 ± 7 to 90 ± 6 ms).

View larger version (17K): [in this window] [in a new window]   Fig 1. Representative monophasic action potentials. These recordings were taken from a control heart, which received no preconditioning. The action potential duration shortens during acute regional ischemia(Ischemia) and returns to baseline during reperfusion(Reperfusion).

View larger version (14K): [in this window] [in a new window]   Fig 2. The incidence of ventricular fibrillation during reperfusion of anterior descending artery and the first obtuse marginal artery (LAD) is indicated as a fraction of the number of experiments performed. *p < 0.05 with respect to control.

Regional LAD occlusion significantly reduced systolic pressure (control, p < 0.001; IPC, p < 0.001; amrinone, p = 0.034). This change was immediately apparent, plateauing within 15 minutes and remaining unchanged until the end of the ischemic period. At the 60-minute time point there was no significant difference among the groups (control = 71 ± 5 mm Hg; IPC = 56 ± 7 mm Hg; amrinone = 65 ± 13 mm Hg). During reperfusion systolic pressure did not recover to preischemic values; no differences among the groups could be appreciated (control = 80 ± 3 mm Hg; IPC = 80 ± 5 mm Hg; amrinone = 81 ± 4 mm Hg).

Diastolic function
As seen in Figure 3 , end diastolic pressure was adversely affected by ischemic preconditioning, rising from 10 ± 1 to 16 ± 2 mm Hg (p = 0.53). Amrinone preconditioning had no effect (7 ± 1 to 8 ± 1 mm Hg). Throughout the ischemic period, diastolic pressure remained unchanged and not demonstrably different than before regional ischemia. By the end of reperfusion, however, diastolic pressure had risen significantly in the control (10 ± 1 to 25 ± 4 mm Hg, p = 0.05) and IPC (10 ± 1 to 23 ± 2 mm Hg, p = 0.001) hearts. Diastolic pressure was maintained at preischemic levels in amrinone hearts (7 ± 1 to 16 ± 4 mm Hg, p = 0.60).

View larger version (15K): [in this window] [in a new window]   Fig 3. Changes in end diastolic pressure during preconditioning, regional ischemia, and reperfusion. Ischemic preconditioning was conducted from -20 to 0 minutes and amrinone preconditioning from -10 to 0 minutes. Regional ischemia in the anterior descending artery and the first obtuse marginal artery (LAD) took place from 0 to 60 minutes and reperfusion from 60 to 120 minutes. *p < 0.05 with respect to control hearts. (IPC = ischemic preconditioning, Am = amrinone preconditioning.)

During reperfusion, peak pressure remained depressed in all groups, mainly as a result of a loss of systolic pressure combined with a rise in diastolic pressure. In amrinone hearts, however, peak pressure was better preserved, as diastolic pressure did not increase in this group. In fact, in amrinone hearts there was no significant difference in peak pressures recorded during reperfusion compared to those seen after preconditioning (72 ± 8 mm Hg at t = 75' [p = 0.293], and 66 ± 5 mm Hg at t = 120' [p = 0.116]).

Effect of preconditioning on infarct size
In control hearts, mean infarct size was 44% ± 4% of the area at risk (Fig 4). Amrinone and ischemic preconditioning both reduced infarct size (amrinone: 26% ± 6%, p = 0.05; IPC: 31% ± 4%, p = 0.10).

View larger version (15K): [in this window] [in a new window]   Fig 4. Infarct size is presented as a percentage of the left ventricular area at risk (see text for detailed calculation). *p < 0.05 with respect to control hearts.

	Comment

Top Abstract Introduction Material and methods Results Comment References

Although not quite reaching significance, there was a trend for the action potential in preconditioned hearts to be prolonged beyond the action potential observed in control hearts. This suggests that preconditioning may prolong the effective refractory period of cardiac muscle. This would be expected to augment the phenomenon of postrepolarization refractoriness commonly observed in cardiac myocytes recovering from transient ischemia [18–21]. Although we did not specifically measure refractory periods in this preparation, any increase in cellular refractoriness would be expected to help prevent common reperfusion arrhythmias, particularly those of a reentrant nature. It is important to note, however, that many patients undergoing cardiac surgery receive sympathomimetic drugs or digoxin. When these drugs are combined with a prolongation of the action potential, there is an increased incidence of triggered arrhythmias such as early or delayed afterdepolarizations.

With respect to myocardial function, ischemic preconditioning did not preserve either peak or diastolic pressures. In fact, ischemic preconditioning exerted an adverse effect by decreasing systolic and peak pressures and increasing end diastolic pressure (EDP) even before regional ischemia. Amrinone preconditioning, in contrast, preserved systolic pressure before regional ischemia and diastolic pressure throughout the experiment. This led to a greater recovery of peak pressure in amrinone hearts. This corroborates work by other investigators, who have shown that amrinone-infused postreperfusion enhances recovery of global function in ischemic myocardium [14]. Both ischemic and amrinone preconditioning reduced the percentage of the left ventricle that was infarcted.

Putative mechanisms of preconditioning
As measured by reduction in infarct size, there are many agents that elicit a preconditioning response. Transient regional and global ischemia, -adrenergic agonists [4], adenosine [8], adenosine triphosphate-sensitive potassium channel openers [9], phorbol esters, morphine, and rapid overdrive pacing [2] have all been studied. The intracellular mechanisms by which preconditioning exerts its protective effect have not yet been clarified, although it appears that protein kinase C probably plays a pivotal role for most of them.

Because many of these drugs are known to interact with at least one member of the family of G proteins, which in turn modulate the activity of the adenylate and guanylate cyclases, it is reasonable to expect that alterations in the intracellular concentration of cAMP and perhaps 3', 5'–cyclic guanosine monophosphate (cGMP) play a role in preconditioning. In general, cAMP levels rise during stress. Preconditioning appears to attenuate that rise [2, 11]. Perhaps raising cAMP levels before an ischemic insult stimulates feedback mechanisms that go on to reduce cAMP levels during stress [12]. By inhibiting phosphodiesterase, amrinone preconditioning may increase the level of intracellular cAMP, preparing the cell against a future ischemic insult.

Clinical applications
With the current surge in interest in minimally invasive techniques for coronary revascularization, there has appeared a need for an effective myoprotectant. Currently practiced techniques of minimally invasive direct coronary artery bypass grafting have employed transient ischemia of the left anterior descending coronary artery in an attempt to produce preconditioning. As we have demonstrated in this study, ischemic preconditioning is not without its deleterious side effects. As suggested by these data, amrinone, a commonly employed inodilator, may serve to replace transient ischemia as an effective preconditioning agent for the heart undergoing minimally invasive direct coronary artery bypass grafting.

Study limitations
This study has established that amrinone preconditioning protects the regionally ischemic heart at least as effectively as ischemic preconditioning. It does not, however, provide any data concerning the underlying mechanism. Because we did not directly measure intracellular cAMP, we cannot determine over what time course the concentration of cAMP might have been altered. This remains a topic for further investigation.

Furthermore, because the isolated perfused heart is not subject to reflex inputs from an intact organism, it is impossible to predict what effect this dose of amrinone will have on systemic vascular tone and sympathetic activity, and how those changes will influence the heart. In addition, because we delivered amrinone directly to the aortic root of an isolated heart in an asanguinous model, and not through a vein in an intact animal, it is not known what role systemic metabolism and distribution play. Studies on intact animal preparations will be required to address these issues.

Finally, although we demonstrated a reduction in the occurrence of ventricular fibrillation during reperfusion in this model, we did not quantify this phenomenon. By attempting to induce fibrillation in each heart, and thereby establishing a fibrillation threshold, we might have been able to better display amrinone’s protective effect.

In this communication we have demonstrated that administration of the phosphodiesterase inhibitor amrinone results in preconditioning of the heart against subsequent ischemic damage. We have called this amrinone preconditioning. Taken together, the data discussed here strongly suggest that amrinone preconditioning provides protection from regional ischemia and reperfusion that is at least as effective as that provided by ischemic preconditioning, without most of its deleterious effects.

	Acknowledgments

 
This work was supported in part by National Institutes of Health grant R29-HL48751.

	References

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