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Ann Thorac Surg 2000;70:602-608
© 2000 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, University Hospital at Stony Brook and the State University of New York, Stony Brook, New York, USA
b Division of Cardiothoracic Surgery, Beth Israel Deaconess Hospital and Harvard Medical School, Boston, Massachusetts, USA
Address reprint requests to Dr Krukenkamp, Division of Cardiothoracic Surgery, T19, 080 Health Sciences Center, Stony Brook, NY 11794-8191
e-mail: ibkmd{at}hotmail.com
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Abstract |
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Methods. Thirty-seven sheep were divided into seven groups; animals received no pretreatment, pinacidil, pinacidil and potassium channel opener blocker glibenclamide, protein kinase C activator 4ß-phorbol-12,13-dibutyrate (PDBu), or PDBu and protein kinase C blocker chelerythrine. The last two groups underwent opposite blockade, chelerythrine + pinacidil, or glibenclamide + PDBu. All groups underwent 60 minutes of regional ischemia followed by 180 minutes of reperfusion. Regional function was assessed throughout the experiment, and at the conclusion of the study the infarct size (as a percentage of the area at risk) was determined.
Results. Infarct size decreased in the groups receiving only pinacidil or PDBu (control: 54% ± 3%, pinacidil: 25% ± 2%, PDBu: 21% ± 3%; p < 0.05 pinacidil or PDBu versus control). This preconditioning protection was lost when the direct blocker was given (58% ± 5%, glibenclamide + pinacidil; 70% ± 6%, chelerythrine + PDBu; p = not significant versus control). The preconditioning response was again attenuated when the opposite blockers were given (64% ± 5%, chelerythrine + pinacidil; 63% ± 1%, glibenclamide + PDBu; p = not significant versus control). There was no significant difference in regional function.
Conclusions. This study shows that both protein kinase C and potassium channels are necessary and codependent for preconditioning in the in vivo heart.
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Introduction |
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-adrenergic stimulation [5], protein kinase C (PKC) stimulation [6], and ATP-sensitive potassium channel openers [7]. Other researchers have also shown the possibility of preconditioning arising from the stress associated with cardiopulmonary bypass [8]. All investigators show a significant myoprotective result, usually based on infarct size reduction. Combining preconditioning with other myoprotective agents such as cardioplegia has been postulated to increase the viability of tissue undergoing ischemia [9]. Although many investigators have been able to reproduce the preconditioning response, the intracellular signaling pathways have yet to be fully understood. Extracellular stimuli are believed to initiate an intracellular process involving G proteins, diacylglycerol, PKC, and possibly the ATP-sensitive potassium channels.Speechly-Dick and colleagues [10] have shown a preconditioning response in human atrial tissue where the ATP-sensitive potassium channel and PKC were both involved. However, they did not describe the relationship between the ATP-sensitive potassium channel and PKC.
Therefore, this study was developed to determine the independent and dependent contributions of the ATP-sensitive potassium channels and PKC toward the development of cardiac preconditioning.
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Material and methods |
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Surgical preparation
Thirty-seven Dorsett or Suffolk sheep (35.0 to 45.0 kg) of either sex were sedated with intramuscular ketamine (20 mg/kg) and anesthetized with intravenous pentobarbital (25 mg/kg). A tracheostomy was performed and ventilation begun with a respirator. The right external jugular vein and common carotid artery were cannulated for intravenous access and arterial pressure monitoring (Millar Instrument, Houston, TX). Animals were heparinized (250 USP/kg) and paralyzed with pancuronium (50 µg/kg). Lidocaine (1.5 mg/kg intravenous bolus, 1 mg/min infusion) and procanamide (12 mg/kg bolus, 3 mg/min infusion) were administered for antiarrhythmia prophylaxis. A partial sternectomy with bilateral anterior rib resection was performed. The first or second diagonal branch of the left anterior descending artery was atraumatically isolated and snared to define a regional area measuring between 5% and 12% of the mass of the left ventricle. Two pairs of ultrasonic transducer crystals (Iowa Doppler Products, Iowa City, IA) were placed (one pair parallel and one pair perpendicular to the epicardial fibers) around the expected bounders of the area at risk. A 5F pressure transducer (Millar Instrument) was inserted through the left ventricular apex to measure intracavitary pressure.
Data acquisition and experimental protocol
Animals were subdivided into seven groups. A group of 6 animals served as nonpreconditioned controls and received 60 minutes of normothermic left anterior descending artery ischemia, followed by 150 minutes of reperfusion. Group 1 animals (n = 5) underwent a 5-minute infusion of pinacidil (Pin) (Research Biochemicals International, Natick, MA) at 30 µmol/kg body weight before left anterior descending artery ischemia. Group 2 animals (n = 5) received 5 minutes of 4ß-phorbol-12,13-dibutyrate (PDBu) (Sigma Chemical Company, St. Louis, MO) at 1 µmol/kg body weight. Group 3 animals (n = 5) received 5 minutes of glibenclamide (Glib) (Sigma Chemical Company) at 1 mg/kg body weight for 5 minutes, immediately followed by Pin. Group 4 (n = 5) received 5 minutes of chelerythrine (Chel) (Sigma Chemical Company) at 9.4 mg/kg body weight, immediately followed by PDBu. Group 5 (n = 5) received 5 minutes of Chel followed immediately by 5 minutes of Pin, and group 6 (n = 6) received 5 minutes of Glib immediately followed by 5 minutes of PDBu. Using a constant infusion pump, all drugs were injected into the left atrium.
At the conclusion of the protocol, the previously occluded diagonal branch of the left anterior descending artery was ligated and monastryl blue pigment (Engelhard Corporation, Louisville, KY) was instilled in the aortic root to delineate the nonstained perfusion bed. Hyperkalemic cardioplegia was infused into the aortic root and hearts were excised and trimmed of right ventricular free walls, atria, chordae tendineae, and valvular tissue. The remaining left ventricle was sliced transversely into approximately 1-cm thick sections and weighed. Nonischemic areas, stained with monastryl blue, were sharply demarcated from dye-free areas. Both sides of each slice were traced onto an acetate sheet to define the area at risk (AR). All slices were incubated in phosphate-buffered 3,5,5-triphenyletrazolium chloride (Sigma Chemical Company) at 38°C for 15 minutes. Viable tissue, stained red, was distinguished from pale necrotic myocardium. Both sides of each slice were again traced to determine the areas of infarct. Left ventricular areas, AR, and infarct areas were planemeterized with the aid of a digitizing graphic tablet (Summagraphics, Seymour, CT). Infarct area (IA) and AR was calculated based on the following formula: weight of infarct (or risk) = weight of slice x (infarct area(side 1)/left ventricular area(side 1)) + (infarct area(side 2)/left ventricular area(side 2))/2. Results are expressed as AR as a percentage of IA.
The percent change in regional area was recorded before and after any intervention, and every 15 minutes throughout the protocol. This was calculated as [(end-diastolic regional area - end-systolic regional area)/(end-diastolic regional area)] x 100%. All data were acquired for 7.5 seconds and digitized at 500 Hz with the use of a 486 processor personal computer (Dell Corp, Austin, TX). The data was stored on the computer for future analysis, which was accomplished with software developed in this laboratory.
Statistical analysis
Data are expressed as mean ± standard error of the mean. For between group comparisons of variables, a one-factor analysis of variance was used (Systat, Evanston, IL). When the value was found to be significant, pair-wise comparisons were made using Tukey’s post hoc analysis. Differences were considered significant when p value was less than 0.05.
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Results |
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Regional function
Regional mechanical function was assessed by sononmicrometry. Percent change in area did not discriminate the preconditioning state (Figs 4 and 5). Percent area change decreased in all groups with ischemia. With reperfusion, mechanical function tended to increase as compared to ischemic function in some groups, but did not fully recover in any group to pretreatment values, suggesting the hearts were stunned by the hour-long ischemia. Figure 4 displays the changes in Pin preconditioning and the effect of blockers infused before Pin. Figure 5 displays similar changes with PDBu preconditioning. Postmortem analysis determined that the ultrasonic transducers were completely in or within 1 cm of the area at risk.
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Comment |
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A common theory is that the potassium ATP channel is stimulated by PKC, which is stimulated by a G protein. If the potassium ATP channel is at the end of the preconditioning cascade, the stimulating the potassium ATP channel while blocking PKC should produce a preconditioning effect. In our model, stimulation of the potassium ATP channels or PKC was sufficient to produce the myoprotective effect of preconditioning. This is similar to results from many other groups. Also, blocking the potassium ATP channel before stimulation of PKC eliminated the preconditioning effect. This is in agreement with several recent studies [10, 15, 16]. However, this is the first study to block PKC activation before stimulating the potassium ATP channels. We showed that blocking PKC with Chel before stimulating the potassium ATP channels blocked preconditioning. This result combined with Glib blocking preconditioning with activation of PKC, means that both PKC and the potassium ATP channels need to be working to produce the preconditioning effect. Therefore, this study is the first to determine that the relationship between the potassium ATP channel and PKC is not a linear cascade, but rather a codependent relationship.
Potassium ATP channel openers are clinically used a vasodilators, which could explain the decrease in left ventricular developed pressure in the Pin group. However, in the groups with either the potassium ATP channels blocked or PKC inhibited, the decrease in developed pressure after Pin was somewhat surprising. The dose of Glib used was sufficient to block the infarct size reduction commonly associated with preconditioning, but was not sufficient to reverse the vasodilatory effects of Pin. Steinberg and colleagues [17] showed that Pin may have a larger affinity for vascular smooth muscle cell receptors stimulating vasodilatation, than for receptors stimulating the preconditioning response. A drug with a higher affinity for myocardial potassium ATP channels would not produce as much peripheral vasodilatation; however, such compounds are still in the developmental stage [17, 18]. When Chel was given before Pin, this decrease in developed pressure was again observed. The role of PKC in vascular smooth muscle cells may be different than its role in the myocyte, where Chel did block the protective effect of Pin. Infusion of Pin decreased developed pressure in all groups receiving Pin, regardless of whether a blocker was infused before Pin. The change in regional area also decreased after infusion of Pin; however, this decrease was not present if either Glib or Chel was infused before Pin. We did not, however, measure coronary blood flow, which would have provided direct evidence of the degree of coronary vasodilation. The decrease in developed pressure caused a decrease in systemic pressure, which may have an effect on preconditioning. The role of systemic hypotension yet remains unclear as all three groups receiving Pin displayed a decrease in pressure, but there were different outcomes between the groups, minimizing the effect which hypotension may have played.
Pharmacologic preconditioning has been shown to be an effective means of protecting the heart from prolonged ischemia. Before it is adapted to clinical use, however, the effect of different blockers (receptor and pharmacologic) must be determined. Preconditioning a patient with a potassium ATP channel opener who is on gliburide (Glib) may not be very useful. Recently, Cleveland and coworkers [19] were unable to precondition isolated human atrial trabeculae from patients who were on oral sulfonylureas. Therefore, it is important to determine the end effector in the preconditioning cycle. If the potassium ATP channel is further downstream in the cycle than, for example, the ß-receptor, then this group of patients may benefit from preconditioning with potassium channel openers, where patients treated with oral sulfonylureas may not benefit from this type of pharmacologic preconditioning.
Two major problems with ATP-sensitive potassium channel openers are their systemic hypotension and increased heart rate effects. Therefore, it is important to give the drugs (especially Pin) as close to the coronary arteries as possible, to minimize any systemic metabolism that might occur. In this study, Pin, PDBu, Glib, and Chel were all given into the left atrium. The optimal point of infusion would be intracoronary. The intraatrial doses where still small enough to have minimal systemic effect, yet still large enough to produce the desired preconditioning effect from Pin and PDBu.
Although other investigators have used prolong ischemic times from 30 minutes through 2 hours, we selected 1 hour of regional ischemia as a clinically relevant time period. An example of this might be during a failed intracoronary intervention that results in ischemia until a surgical intervention can be achieved. In fact, 1 hour of ischemia was substantial to produce a significant difference in infarct size between preconditioned and nonpreconditioned groups.
We also believe that 30 minutes of global ischemia, as used by many investigators, may not be clinically relevant. It is likely that if a patient experience 30 minutes of global myocardium ischemia, there has been a circulatory arrest. The heart may be able to recover, but other systemic organs (ie, brain, kidney) may not be as fortunate. Although 30 minutes of global ischemia may be experienced during cardiac operations, the clinical effect of preconditioning and cardioplegia is still controversial [20].
A clinical situation in which ischemic preconditioning may be effective is during minimally invasive direct coronary artery bypass. During this procedure, a region of the heart is made ischemic to allow for a bloodless operative field. In difficult situations, ischemic times easily exceed 30 minutes and may approach 1 hour.
Limitations of this study
Mechanical function was accessed by changes in end-diastolic regional area. Unfortunately, this method cannot discriminate between stunned myocardium and infarcted myocardium, as we have previously demonstrated [8]. To properly determine whether the myocardium is stunned and not infarcted, reperfusion for 24 hours would be necessary [21]. Therefore, lack of evidence for differences in mechanical function may actually obscure the percentage of stunned versus infarcted myocardium in the preconditioned groups compared to the nonpreconditioned groups. Also, because of biological variance, the AR differs in each animal, making placement of the ultrasonic transducers in relation to the AR difficult. Although the transducers were within the AR for all the animals, any border zone (region between ischemic and nonischemic zones) effects could add to the variance in the measured changes in myocardial function. Our laboratory is currently developing a technique that will be able to determine deformation in the principle direction [22]. This technique will also be able to determine whole field deformation with high spatial resolution, which will reduce the inherent problem of locating the transducer within the AR. Also, the axes on which the fibers lay varies through the thickness of the myocardium [23] and therefore, the exact orientation of the transducers cannot be accurately determined, which may result in an underestimation of the deformation.
Horneffer and coworkers [24] report that measuring infarct size with triphenyl-tetrazolium chloride is accurate and reproducible between 2 to 48 hours of reperfusion as compared to histology in swine. In pilot animals we found no difference in infarct size, as measured by triphenyl-tetrazolium chloride staining, at 2 or 3 hours of reperfusion. Early triphenyl-tetrazolium chloride staining can underestimate the infarct size if drugs that inhibit the washout of the dehydrogenase enzyme are given during reperfusion [25]. No drugs that are known to effect enzyme washout were given during reperfusion.
Although all animals received lidocaine and procanimide to help prevent the occurrence of arrhythmias, in some animals (10%), ventricular fibrillation still occurred. However, because the absolute numbers are so small, a much larger sample size would be needed to determine whether any differences exist between groups. It is interesting to note that all animals with sustained ventricular fibrillation were from the control group or a group with a blocked preconditioning response.
Ischemic preconditioning requires a reperfusion phase before the prolonged ischemic event. Whether pharmacologic preconditioning also requires this reperfusion or drug washout phase is still unknown. In pilot studies in our laboratory, we found that this washout period was not required to generate the protective response of preconditioning with Pin in the rabbit heart. However, it is not known whether this response is conserved in all species. It should be noted that in this study a drug-free washout period did not occur before the prolonged ischemic insult. Whether a pharmacologic agent infused before a prolonged ischemic insult should be termed pretreatment rather than preconditioning is debatable; however, it was referred to as preconditioning in this study.
Clinical relevance
Although clinically we are not yet at the point of infusing pharmacologic agents solely to generate a protective preconditioning response, ischemic preconditioning is being used clinically in minimally invasive and beating heart operations. However, the inherent effects of additional ischemic time may not be helpful in all patients, especially if the time required to construct the anastomosis is of a similar time duration as that used in ischemic preconditioning. Also, it is important to note that patients receiving glyburide, an ATP-sensitive potassium channel blocker, may not benefit from ischemic preconditioning. Our results suggest that it is important for both the PKC pathway and the ATP-sensitive potassium channels to be functional, which is important information in developing the optimal pharmacologic agent to be used clinically to protect the heart.
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