Does Cardiopulmonary Bypass Alone Elicit Myoprotective Preconditioning?
Background Brief episodes of ischemia can precondition myocardium. Ischemic preconditioning (PC) has been proposed as an adjuvant method of improving myocardial protection during cardiac surgery. It is unknown whether CPB without an episode of ischemia generates the PC response.
Methods and Results To prove that PC occurs in sheep, groups 1 (non-CPB control) and 2 (non-CPB ischemic PC, three 5-minute episodes of normothermic regional ischemia) were studied. Groups 3 (CPB alone), 4 (CPB–alpha receptor blockade, phentolamine 5 mg/kg), and 5 (CPB–adenosine receptor blockade, 8-sulfophenyltheophylline 5 mg/kg) were placed on CPB for 30 minutes and subsequently weaned. All groups underwent 60 minutes of normothermic regional ischemia and 150 minutes of reperfusion. The area at risk (AR) was delineated by Monastryl blue pigment, whereas the infarct size (IS) was determined by tetrazolium staining. Body mass, left ventricular mass, and AR were not different between groups. Ischemic PC was demonstrated in this ovine model by a 54% reduction of IS relative to AR (group 1 versus group 2, P<.01). CPB alone produced a similar percentage IS reduction without ischemia (group 3 versus group 1, P<.01) that was prevented by either α-adrenergic receptor (group 4 versus group 3, P<.01) or adenosine receptor (group 5 versus group 3, P<.01) blockade.
Conclusions CPB alone appears sufficient to elicit the PC response important for myocardial protection during cardiac surgery. These data suggest that myocardial α-adrenergic receptor and adenosine receptor stimulation are involved in initiating CPB-induced PC.
Short periods of ischemia separated by intermittent reperfusion render myocardium more tolerant to subsequent ischemic episodes and induce a marked reduction of resultant IS.1 This myoprotective response has been demonstrated in a wide variety of species, including dogs,1 2 3 pigs,4 rabbits,5 6 rats,7 8 9 and sheep10 and has been proposed to exist in humans.11 The PC phenomenon suggests that transient stresses (eg, ischemia, hypoxia,12 heat shock,13 α1-adrenergic stimulation14 15 ) induce intrinsic changes within myocardium to enhance resistance to subsequent ischemia-reperfusion injury. This myoprotective response is effective in reducing a range of postischemia-reperfusion events, including IS and incidence of arrhythmias. The duration of protection appears to be species dependent but generally disappears 1 to 3 hours after the triggering stimulus.15 The precise mechanism by which PC protects the heart, however, remains unknown.16 Present theories include stimulation of one or more types of receptors: adenosine A1 receptors,5 muscarinic receptors,17 or α sympathomimetic receptors.15 18 Working through inhibitory G proteins19 such receptor stimulation may result in translocation of protein kinase C to the cellular membrane. Subsequent phosphorylation of the ATP-dependent potassium channel may be responsible for triggering the PC response.20 21
If the PC response exists in humans, its stimulation may improve myocardial protection during interventional cardiac procedures. To be clinically applicable, this response would have to be stimulated through either pharmocological or benign physiological stimuli. The present study tested the hypothesis that the endogenous physiological changes that occur with CPB are sufficient to trigger the PC response in a laboratory model of ovine PC.
Animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985).
Twenty-eight Dorsett or Suffolk sheep (36.0 to 47.8 kg) of either sex were sedated with ketamine (20 mg/kg IM) and anesthetized with pentobarbital (25 mg/kg IV). A tracheostomy was performed and ventilation begun with a volume-cycled ventilator (Harvard Apparatus). The right external jugular vein and common carotid artery were cannulated for intravenous access and arterial pressure monitoring (Millar Instruments). A partial sternectomy with bilateral anterior rib resection was performed. The second or third diagonal branch of the left anterior descending artery (LAD) was atraumatically isolated and snared to define a regional area measuring between 5% and 10% of the mass of the LV. Animals in three of the groups were heparinized (250 USP/kg) and instrumented for (CPB) via a single right atrial cannula with an arterial flow rate of 75 mL · kg−1 · min−1 delivered through the left axillary artery. Both ventricles were decompressed via cannulae placed in the pulmonary artery and the LV apex. Attention was paid to the stabilization of arterial blood gases, acid-base status, temperature, hemoglobin level, and electrolyte levels throughout the protocol (pH 7.35 to 7.45; PO2 >100 mm Hg; temperature 37°C; Hgb >5.0 mg/dL; K+ <5.0 mEq/L).
Data Acquisition and Experimental Protocol
Animals were divided into five groups. Group 1 (n=6) animals served as non-preconditioned controls and received 60 minutes of normothermic regional ischemia, followed by 150 minutes of reperfusion. In group 2 (n=6), animals were preconditioned with three 5-minute episodes of normothermic regional ischemia before 60 minutes of sustained normothermic regional ischemia. Groups 3 (n=6), 4 (n=5), and 5 (n=5) were placed on CPB for 30 minutes and were subsequently weaned. Group 3 served as CPB controls and received 60 minutes of normothermic regional ischemia after being taken off CPB for 5 minutes, followed by 150 minutes of reperfusion. Groups 4 and 5 underwent the same protocol but received either nonspecific α-adrenergic–receptor blockade (phentolamine, 5 mg/kg) or adenosine-receptor blockade (8- SPT, 5 mg/kg) before the initiation of CPB. Lidocaine (2 mg/kg IV bolus) was administered for anti-arrhythmia prophylaxis prior to the initiation of 60 minutes of normothermic regional ischemia and at the time of subsequent reperfusion.
At the conclusion of the protocol, the previously occluded diagonal branch of the LAD was ligated and monastryl blue pigment was instilled in the aortic root to delineate the nonstained perfusion bed. After hyperkalemic cardioplegia, hearts were excised and trimmed of right ventricular free walls, atria, chordae tendineae, and valvular tissue. The remaining LV were transversely sliced into 1-cm-thick sections and weighed. Nonischemic areas, stained with monastryl blue, were sharply demarcated from dye-free. Both sides of each slice were traced onto an acetate sheet to define the AR. All slices were incubated in phosphate buffered 3,5,5-triphenyltetrazolium chloride at 38°C for 40 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. LV areas, AR, and infarct areas were planimetered with the aid of a digitizing graphic tablet. IS was calculated with the following formula: weight of infarct=weight of slice×(infarct area[side 1]+infarct area[side 2])/(LV area[side 1]+LV area[side 2]).
In selected animals, regional mechanical function was assessed using sonomicrometry. Two orthogonal pairs of dimension crystals were arranged both parallel and perpendicular to fiber shortening within the ischemic zone. Percent regional area change was recorded every 30 minutes throughout the study protocol. This was calculated as [(end diastolic regional area−end systolic regional area)/end diastolic regional area]×100%. In these animals, IS was not determined because of crystal placement.
Data are expressed as mean±SD. For between-group comparisons of hemodynamic variables (as well as body mass, LV mass, AR, and IS), a one-factor ANOVA was used. 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<.05.
Hemodynamic data for the CPB groups (groups 3, 4, and 5) are summarized in Table 1⇓. Heart rate remained stable throughout the protocol in all groups. Mean arterial pressure slowly decreased during the period of sustained regional ischemia and the 150-minute period of reperfusion; however, there were no between CPB–group differences. Groups 1 and 2 maintained a similar heart rate (121±4 beats per minute) while also maintaining mean arterial pressures of ≥50 mm Hg during ischemia and reperfusion.
No animals in either group 1or 2 had ventricular fibrillation, and neither did group 3 (CPB alone). However, two animals in group 4 (CPB+phentolamine) and one in group 5 (CPB+SPT) fibrillated during reperfusion after the 60-minute period of regional ischemia. All animals responded to electrical cardioversion.
AR and IS
Body weight, LV weight, and AR are tabulated in Table 2⇓. IS and the percentage of AR that infarcted are displayed in Table 3⇓. Body weights and LV weights were not significantly different among the five groups. AR were also not significantly different among the groups (10±5 g for group 1 versus 7±3 g for group 2, 7±2 g for group 3, 10±3 g for group 4, and 9±4 g for group 5). The percentage of the risk region infarcted (IS/AR) was 54±6% for group 1, 48±3% for group 4, and 53±9% for group 5. These values were not statistically different from each other. IS of group 2 (25±4%, P<.01 versus groups 1, 4, and 5) and IS of group 3 (18±3%, P<.01 versus groups 1, 4, and 5), which received either short episodes of regional ischemia or CPB alone, were significantly smaller (Fig 1⇓).
Regional Functional Studies
Regional mechanical function assessed by sonomicrometrically determined percent area change did not discriminate the preconditioned state. Fig 2⇓ illustrates the time course of ischemia and reperfusion for animals preconditioned (CPB alone) and not preconditioned (CPB+phentolamine). In both cases, there was systolic bulging as evidenced by a negative percent area change during the ischemic interval. On reperfusion, both demonstrated recovery of systolic function to a hypokinetic or akinetic, but not dyskinetic, level. There was no difference during the time course of reperfusion between these studies.
We have previously described a significant reduction of IS in ovine myocardium with ischemic PC, a response that is well preserved in aged sheep.10 In the present study, we both confirmed the effectiveness of ischemic PC in reducing myocardial IS and investigated the ability of total CPB to trigger the PC response in this ovine model. Our results demonstrate that CPB is as potent a stimulus as three brief periods of ischemia for initiating the PC response, as measured by a reduction in IS. α-Adrenergic–receptor or adenosine-receptor blockade prior to the initiation of CPB prevented the PC response.
Although the precise mechanism of ischemic PC is still under investigation, several theories have been discounted in the past 8 years. Ischemic PC does not appear to be related to recruitment of collateral flow, stunning, acute protein synthesis, inhibition of mitochondrial adenosine triphosphatase, or protection by antioxidants.16 One presently favored explanation is the adenosine hypothesis. Adenosine, which is released during ischemia-reperfusion injury, stimulates adenosine A1 receptors, which couple to inhibitory G proteins.19 Through second-messenger pathways, protein kinase C is translocated from the cytosol to the myocyte membrane. This results in the phosphorylation of an unidentified protein that in turn may mediate actual PC. The ATP-dependent potassium channel that becomes activated during ischemic PC may be the responsible mediating protein. Acting through shortened action potential duration and decreased calcium ion influx, an energy-sparing loss of contractile function and preservation of cellular ATP may result.20 21
Another, similar, theory is that α-adrenergic stimulation (α1-receptor mediated) may trigger PC working through a similar inhibitory G-protein mechanism.18 Since myocardial ischemia has been shown to induce the release of both endogenous catecholamines and adenosine, it may be that both α1-adrenergic–receptor and adenosine A1–receptor activation together contribute to ischemic PC in some species.
CPB has many complex effects on both myocardial and systemic end-organ perfusion. The loss of atrial and ventricular filling may stimulate a sympathetic-receptor mediated release of local catecholamines, whereas the interruption of pulsatile systolic and diastolic blood flow to the adrenal glands may stimulate a systemic catecholamine release. Therefore, an altered adrenergic state may be partially responsible for CPB-associated PC in ovine myocardium. Ischemia and hypoxia were controlled for in our model by ensuring sufficient systemic perfusion and by careful monitoring of animal pH, arterial PO2, and hemoglobin. Banerjee et al15 observed in isolated rat hearts that norepinephrine or phenylephrine triggered PC, measured by recovery of LV function, and that this triggering was prevented by adrenergic blockade with reserpine pretreatment. They concluded that the beneficial effects of ischemic PC were mediated by release of neurotransmitters and stimulation of α1-adrenergic receptors. Similarly, Thornton et al22 demonstrated that tyramine, an agent that causes release of endogenous catecholamines, reduced IS in rabbits when given prior to a sustained period of ischemia. Furthermore, this effect was not evidenced after α-adrenergic–receptor blockade but was seen after β-adrenergic–receptor blockade. In our ovine model, CPB-associated PC was not evident after α-adrenergic–receptor blockade.
Adenosine is released in large quantities within seconds after the onset of myocardial ischemia23 and after catecholamine stimulation.24 Adenosine has been demonstrated to be involved in ischemic PC in many species. In the canine model, Gross and Auchampach21 induced protection against infarction with exogenous adenosine. Conversely, the selective adenosine A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine abolished the protective effects of ischemic PC in this same species.21 In our ovine model, CPB-associated PC was blocked by 8-SPT. Therefore, catecholamine-mediated release of adenosine may be involved in triggering CPB-associated PC in ovine myocardium. It is not possible to specify either the configuration of cellular receptors or the specific occupancy responsible for initiating CPB-associated PC. However, it is possible to conclude from these data that an adrenergic-adenosine receptor interaction is necessary.
Potential Model Limitations
Regional mechanical function was difficult to quantify in this preparation given the complex relation between myocardial necrosis and presumed stunning induced by 60 minutes of normothermic regional ischemia in the ovine species with limited native collateral coronary circulation.25 All animals displayed obvious regional systolic bulging during the periods of ischemia. Analysis of mechanical function using regional sonomicrometric crystals in pilot animals during the reperfusion period demonstrated an inability to distinguish the myoprotective benefit of increased myocyte survival due to PC from the associated deleterious effects of regional myocardial stunning. This is not surprising given the complex arrangement of areas of necrosis with areas of stunning and the relation of crystal placement within the zone of interest. Moreover, prior data would indicate that a reperfusion period of >24 hours is required for full recovery of regional mechanical function.26 We must conclude, therefore, that in this model, regional mechanical function did not distinguish the preconditioned state. Interestingly, the myoprotective decrease in arrhythmias associated with PC was seen in group 3 when compared with groups 4 and 5. The small numbers involved precluded statistical analysis of this observation.
IS in this study was determined by staining with triphenyltetrazolium chloride stain. Determination of the extent of early infarction by this method has generally been found to be an accurate measure of ultimate IS at 2 to 48 hours of reperfusion in swine when compared with subsequent histiological analysis in animals not receiving further treatment.27 Tetrazolium staining has been demonstrated to reveal equivalent IS values compared with histological determination in dogs28 and rabbits29 after 2 to 3 hours of reperfusion. Downey and coworkers5 8 19 have routinely used tetrazolium staining following 2- to 3-hour periods of reperfusion after sustained ischemia to extensively investigate the PC phenomenon. In our pilot animals, there were no differences in IS, as measured by tetrazolium staining, after varying periods of reperfusion that extended from 2 to 4 hours. However, early tetrazolium staining has been found to underestimate IS if an intervention occurs that inhibits the washout of enzymes from irreversibly injured myocardium.30 We cannot exclude this potential limit of our experimental model, but no animal in groups 1, 2, or 3 received any drug that is known to affect the washout of tissue dehydrogenase enzymes. The effect of the α-adrenergic–receptor- and adenosine-receptor blockade on dehydrogenase enzyme levels is unknown, but these groups did not undergo any IS reduction when compared with control animals.
PC has been recognized as one of the most powerful and reproducible methods of delaying myocyte necrosis known to date. A wide variety of stresses have been shown to stimulate the PC response in laboratory animals. Since its discovery 8 years ago, the potential clinical applications of this mechanism have received much attention in the literature. The demonstration that CPB alone stimulates PC in sheep provides evidence that this myoprotective response may be clinically initiated during routine cardiac surgery. The duration of such protection is most likely short-lived and limited to the immediate intraoperative period. Further investigation is needed to develop methodology that allows the ongoing stimulation and extension of myoprotective PC into the extended postoperative period. Such techniques would also promise to be clinically relevant to both nonoperative interventional and noninterventional care of coronary artery disease.
Selected Abbreviations and Acronyms
|AR||=||area(s) at risk|
|LV||=||left ventricle(s)/left ventricular|
This study was supported by National Institutes of Health grants R29-HL48751 and HL29077.
Reprint requests to Irvin B. Krukenkamp, MD, Division of Cardiothoracic Surgery, New England Deaconess Hospital, 110 Francis St, Suite 2-C, Boston, MA 02215.
- Copyright © 1995 by American Heart Association
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