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Does localized myocardial [K.sub.ATP] channel opening result in global cardiac instability?

INTRODUCTION

Ischemic preconditioning (IP) refers to an endogenous protective mechanism of the localized myocardial tissues to limit the damage and infarct size of a subsequent ischemic event (1). It is thought to be induced by preceding short periods of ischemia that "precondition" the myocardial tissue through indigenous biochemically-mediated adaptations (1). IP is thought to be mediated by [delta]-opioid receptor stimulation and induction of KATP channel opening (2,3,4). Recent studies have provided evidence that the cardioprotective effects of IP may be systemically mediated, triggered by some humoral compound that can influence distant tissues (1). While this compelling finding has some exciting potential from a therapeutic standpoint, it also raises many new questions that are fundamental to our understanding of the mechanisms of ischemic preconditioning. Is this coronary effluent simply a spillover of the local preconditioning or is this newly discovered "action at a distance" also an important part of the protective mechanism? From both a scientific and medical perspective it is important to ascertain if these global effects are necessary to maintain general cardiac and circulatory integrity. In an effort to answer this question a porcine model was developed in which a segment of myocardium and its coronary supply could be isolated from the remainder of the cardiac tissue. D-Ala2-Leu5-enkephalin (DADLE), a [delta]-opioid receptor agonist known to induce a state of KATP channel opening similar to the mechanisms of IP, was infused into the distal segment of the left anterior descending artery (LAD) during an acute coronary occlusion to examine the effects on overall cardiovascular stability (2). IP through a mechanism of KATP channel opening is cardioprotective and limits local myocardial tissue necrosis. However, when this effect is confined to a limited area of the heart, segmental delays in myocardial repolarization may produce global cardiac electrical instability.

DADLE: IP is thought to be may be mediated by [delta]-opioid receptor stimulation and induction of KATP channel opening (3,4). DADLE is a [delta]-opioid receptor agonist that has been extensively studied with regard to its effect on myocardial cell function and has been used as a tool to investigate the mechanisms of IP (2,5). In fact, [delta]-opioid receptor agonists have been shown to limit infarct size and lower myocardial oxygen consumption (6,7). Additionally, it is thought by some investigators that an endogenous compound similar to DADLE may be the cardioprotective link between preconditioning and natural hibernation in animals (2). These factors make DADLE an excellent tool for simulating a state of localized IP. While [delta]-opioid receptor blockade has been found to have an antiarrhythmic effect during acute coronary occlusions, systemic infusions of DADLE are not arrhythmogenic and have no effects on the electrocardiogram during coronary occlusions (5,8).

MATERIALS AND METHODS

All experiments were conducted in anesthetized male or female pigs weighing 22[+ or -]1 Kg. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center and experiments were conducted according to the Guide for the Care and Use of Laboratory Animals and the guidelines of the Animal Welfare Act.

Approximately 15 minutes prior to anesthesia, all pigs were administered atropine (0.025mg/kg i.m.), meditomidine (0.08mg/kg i.m.), and butorphanol (0.2mg/kg i.m.). Anesthesia was induced with ketamine (10mg/kg i.m.), the pigs intubated, and anesthesia maintained with isoflurane. An ear vein was cannulated and saline infused at 10 ml/kg/hour. A femoral artery was catheterized and used for measuring arterial pressure, pulse pressure, heart rate and DP/dt and for sampling systemic arterial blood.

In a restricted myocardial ischemia-reperfusion protocol, a segment of the myocardium and its blood supply were isolated from the remainder of the heart by a ligation of the left anterior descending coronary artery (LAD) just distal to the 1st branch. In the operative procedure, the heart was exposed via a midline sternotomy, the pericardium retracted, and the LAD isolated and dissected free of surrounding tissues approximately 1/3 from the apex. A silastic tube was placed under the LAD and positioned to allow free flow of blood in the coronary. A 20-gauge needle was placed in the non-occluded LAD just distal to the ligature and perfused with heparinized whole blood at the very slow rate of 1 ml/min so as to limit the quantity of the perfusate released into the general circulation.

The animals were allowed to recover from surgery for at least 30 minutes and until the measured hemodynamic variables had reached a stable baseline, and then baseline control data were collected for 20 minutes. The LAD then was occluded by retracting the silastic tubing and placing a bulldog clamp on the LAD immediately proximal to the tubing. A 20-gauge needle was placed into the lumen of the LAD immediately distal to the occlusion, and 10 ml heparinized whole blood, with (Dpigs) or without (Cpigs) DADLE (Peninsula Laboratories Inc.; 1 mg/kg estimated heart weight) was infused into the LAD over 10 minutes at 1 ml/min. The infusion was stopped, the needle removed, and the occlusion continued for an additional 50 minutes. The occluder and clamp were then removed, followed by a 120-minute reperfusion period or until the animal expired. Reperfusion was determined visually by observing that blood flow to the blanched zone was reestablished. At the end of the reperfusion period, the LAD was reoccluded and a 10 ml bolus of Evan's blue dye infused into the left atrium. The heart was stopped with KCl, removed, and visually examined to verify that only a limited area of the myocardium had been isolated. The hearts were then stained with tetrazolium and infarct size determined with planimetry (9).

During the experiment, the measured hemodynamic variables were sampled at 200 Hz for 12 seconds of each minute, were averaged for this 12-second period, and were collected and digitally converted for analysis by computer. The cumulative average values of these hemodynamic variables were calculated for the different periods for each group (Dpigs and Cpigs) up through the completion of the 10-minute LAD infusion period during LAD occlusion. Data were excluded for analysis during periods of terminal dysrhythmia.

A standard 12-lead EKG was recorded at 25 mm/sec twice during control and several times during the occlusion and reperfusion periods. QT dispersion (QTdisp) was measured with standard technique from the EKG (10). Blinded measurements of the QT intervals (onset of QRS to end of T wave) were determined manually with calipers under magnification (6x). Three consecutive cycles were measured in each of the 12 leads, averaged to obtain a mean QT interval, and corrected for heart rate (R-to-R interval) using Bazett's formula (QTc = QT/[square root of RR]). When the end of the T could not be identified the lead was excluded. A minimum of 6 leads was required for the measurement of QT dispersion. The QT dispersion was calculated as the difference between the minimum and maximum QT intervals within the 12 leads. All episodes of sustained (> ventricular tachycardia (> 30 seconds) or prolonged ventricular arrhythmias requiring cardioversion were recorded. The experiment was terminated after 3 unsuccessful attempts at cardioversion. The relative arrhythmia frequency, degree of QT dispersion, and other hemodynamic variables were compared between groups using Fisher's Exact and t-test (p < 0.05).

RESULTS AND DISCUSSION

In a total of 12 pigs studied (6 Dpigs; 6 Cpigs), 83% of the Dpigs (5 out of 6) and 17% of the Cpigs (1 out of 6) there developed a sustained ventricular tachyarrhythmias within 5 minutes after the infusion of DADLE or sham control into the localized segment of myocardium (Table 1). These dysrhythmias quickly resulted in death of the animal despite repeated attempts at cardioversion. The average QTdisp at the end of the infusion period was found to be 144 msec (95% CI: 107;183) for the Dpigs as compared to 61 msec (35;84) for the Cpigs. When compared to the preocclusion measurements, this represented a 116% increase in the amount of QTdisp over the occlusion period for the Dpigs as compared to only a 22% increase for Cpigs.

The cumulative averages of the different hemodynamic variables over the entire occlusion and infusion period showed stability as compared to their control period average values in the Dpigs (Table 2). However, in the Cpigs a small fall in the mean arterial pressure, pulse pressure and DP/dt were noted during the occlusion and infusion periods. While some of the variations observed are typical of the adrenergic stimulation seen during acute coronary occlusion, these small differences in the percent of deviation from control values were not felt to be hemodynamically destabilizing. Nonetheless, it is important to note that the average DP/dt (an indicator of contractility) fell significantly during the occlusion and infusion period for the Cpigs (-20%) while it remained stable for the Dpigs receiving DADLE during this same period.

Though the tetrazolium staining demonstrated a localized ongoing infarction of myocardium within animals from both groups, the failure to obtain a reperfusion period in 80% of the Dpigs made any reasonable comparisons impossible. However, in those animals that had a sufficient reperfusion period (5 Cpigs, 1 Dpig), infarct size measured an average of 13.8% of the volume of the left ventricle tissue (:5.45;22.15).

DISCUSSION AND CONCLUSIONS

Multiple studies in both the cardiology and emergency medicine literature have established that IP through a mechanism of KATP channel opening is cardioprotective and can limit myocardial tissue necrosis (1,11). The excellent work of Dickson, Przklenk and others has provided evidence that suggests IP is mediated through a humoral factor with the potential for "action at a distance" (1,11). While this finding can have enormous medical implications for the development of therapeutic strategies for patients with acute coronary occlusions, the physiologic significance of this discovery is still a mystery. Mammalian physiology is replete with systems of biochemical messengers that affect feedback and control in response to some stimulus. The majority of these messengers or triggers have very short half-lives and are designed to exert only localized effects within a tissue, organ or vascular bed (i.e. adenosine, cyclic AMP). However, when these blood-borne triggers cause an effect that is systemic or global in nature by an "action at a distance" these compounds fall into the class of biologic substances we call hormones. The fact that IP produces a substance that has the potential of exerting systemic effects requires us to question the physiologic importance of this action if we are to really understand the functioning of the cardioprotective mechanism.

In our study we were initially quite surprised to find that when [delta]-opioid receptor stimulated KATP channel opening (simulating the IP state) is confined to a limited area of the heart, segmental delays in repolarization may produce global cardiac electrical instability. This is in stark contrast to prior evidence demonstrating a lack of arrhythmogenesis and no effect on the electrocardiogram during coronary occlusion when DADLE is infused systemically and has the opportunity for global effects (5). It could be argued that the coronary occlusion and myocardial ischemia was responsible for the arrhythmias seen in our study. However, the overwhelming propensity of the dysrhythmic events to occur after the DADLE infusion, as opposed to the control period, makes that consideration very improbable. In fact, the pigs receiving DADLE were shown to have a preservation of myocardial function (DP/dt) during the occlusion period as compared to the control group despite the comparatively lower survival rate. It is also possible that there could be some washout of the DADLE into the general circulation in spite of the extremely low coronary infusion rate. However, no significant systemic hemodynamic effects of DADLE (bradycardia, hypotension) were noted and the quantity infused was well below that required to maintain physiologically effective blood levels (7). In IP the initial ischemia stimulates production of the humoral substance, which is washed out into the general circulation and prepares the myocardium for the next ischemic event (1). In our model there were no washout periods.

While the baseline mean arterial pressure and pulse pressures were similar for the controls of the two groups, there were significant differences in the control heart rates and DP/dt. It is uncertain what impact this discrepancy might have on the outcomes observed and could be considered a limitation of the study. It is possible that the increased contractility and heart rate also increased the basal myocardial oxygen demand in the DADLE group and resulted in relatively more ischemia during the occlusion period. However, the contractile state was preserved in the DADLE group while the coronary occlusion diminished the cardiac pumping capacity of the control pigs with some hemodynamic instability.

Overall, the findings from this study in conjunction with the previous studies cited suggest that IP is a mixed process with systemic as well as localized mechanisms of action. Examining this phenomenon in light of the evidence presented by Dickson, it is possible to hypothesize that IP must by necessity occur at the systemic level and affects the entire heart and not just the ischemic segment. The myocardium is a complex interplay of electrical and mechanical activity that must function in concert at the both the cellular and organ levels in order for the organism to survive. When ischemia occurs in a localized area of the heart, the segmental cellular elements are forced to down-regulate their metabolic demands and stabilize their membranes in order to mitigate the potentially destructive results of the insult. The heart that is best prepared for this adaptation (i.e. ischemic preconditioning through KATP channel opening) is most likely to survive. Because the electrical as well as the contractile functioning of this ischemic myocardial segment are linked to their metabolic integrity then their functioning also must be impacted. If this results in a change in membrane stability and KATP channel opening occurs only in the localized area it is easy to see how this could cause dispersion in the global electrical events of the heart. Such dispersion in repolarization of greater than ~80 msec is known to result in dysrhythmias and ventricular tachycardia (10,12). If the effects of the DADLE were present within the whole heart then this dispersion would be less likely to develop. Ovize et al. found a shortened time to fibrillation in pigs using a similar occlusion protocol to our study but with natural ischemic preconditioning (13). However, this tendency to fibrillation may not be mediated through changes in the KATP channels (14). There may be other factors related to the [delta]-opioid receptor agonist effect of DADLE that could destabilize the local myocardium. Wu et al. found a reduction in dysrhythmias after coronary artery bypass graft when IP was induced by a protocol that involved a clamping of the aorta (15). This is IP intervention was evidently more global in nature and would therefore have an effect on the entire.

There are several limitations to the study that should be noted. It could be argued that the coronary occlusion and myocardial ischemia was responsible for the arrhythmias seen in our study. However, the overwhelming propensity of the dysrhythmic events to occur after the DADLE infusion, as opposed to the control period, makes that consideration very improbable. In fact, the pigs receiving DADLE were shown to have a preservation of myocardial function (DP/dt) during the occlusion period as compared to the control group despite the comparatively lower survival rate. It is also possible that there could be some washout of the DADLE into the general circulation in spite of the extremely low coronary infusion rate. However, none of the typical systemic hemodynamic effects of DADLE (bradycardia, hypotension) were noted and the quantity infused was well below that required to maintain physiologically effective blood levels (16). In IP, the initial ischemia stimulates production of the humoral substance, which is washed out into the general circulation and prepares the myocardium for the next ischemic event (1). In our model there were no washout periods.

The effect of the physiologic mediators of IP to have an effect globally may be necessary for survival of the organism as a whole and therefore has strong evolutionary pressures for development. It is well recognized that DADLE may be protective against ischemia in a variety of organs other than the heart. It appears that there is still much we do not understand about IP and the area remains an exciting field for investigation (17).

LITERATURE CITED

(1.) Dickson EW, Porcaro WA, Fenton RA, Heard SO, Reinhardt CP, Renzi FP, Przyklenk K. "Preconditioning at a Distance" in the isolated rabbit heart. Acad Emerg Med 2000; 7:311-317.

(2.) Kevelaitis E, Peynet J, Mouas C, Launay JM, Menasche P. Opening of potassium channels: the common cardioprotective link between preconditioning and natural hibernation? Circulation 1999: 99:3079-3085.

(3.) Schultz JE, Hsu AK, Gross GJ. Ischemic preconditioning in the intact rat heart is mediated by delta1--but not mu--or kappa-opioid receptors. Circulation 1998; 97:1282-1289.

(4.) Liang BT, Gross GJ. Direct preconditioning of cardiac myocytes via opioid receptors and KATP channels Circ Res 1999; 84:1396-1400.

(5.) Wong TM, Lee AY, Tai KK. Effects of drugs interacting with opioid receptors during normal perfusion or ischemia and reperfusion in the isolated rat heart--an attempt to identify cardiac opioid receptor subtype(s) involved in arrhythmogenesis. J Moll Cell Cardiol 1990; 22:1167-1175.

(6.) Schultz JE, Hsu AK, Nagase H, Gross GJ. TAN-67, a delta 1-opioid receptor agonist, reduces infarct size via activation of Gi/o proteins and KATP channels. Am J Physiol 1998; 274:H909-914.

(7.) Rosen CL, Cote A, Haddad GG. Effect of enkephalins on cardiac output and regional blood flow in conscious dogs. Am J Physiol 1989; 256:H1651-1658.

(8.) McIntosh M, Kane K, Parratt J. Effects of selective opioid receptor agonists and antagonists during myocardial ischemia. Eur J Pharmacol 1992; 210:37-44.

(9.) Fishbein MC, Meerbaum S, Rit J, Lando U, Kanmatsuse K, Mercier JC, Corday E, Ganz W. Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique Am Heart J 1981;101:593-600.

(10.) Pye M, Quinn AC, Cobbe SM. QT interval dispersion: a non-invasive marker of susceptibility to arrhythmia in patients with sustained ventricular arrhythmias? Br Heart J 1994 Jun;71(6):511-514.

(11.) Przyklenk K, Bauer B, Ovize M, Kloner RA, Whittaker P. Regional IP protects remote virgin myocardium from subsequent sustained coronary occlusion. Circulation 1993; 87:893-899.

(12.) Paventi S, Bevilacqua U, Parafati MA, Di Luzio E, Rossi F, Pelliccioni PR. QT dispersion and early arrhythmic risk during acute myocardial infarction. Angiology 1999;50:209-215.

(13.) Ovize M, Aupetit JF, Rioufol G, Loufoua J, Andre-Fouet X, Minaire Y, Faucon G. Preconditioning reduces infarct size but accelerates time to ventricular fibrillation in ischemic pig heart. Am J Physiol. 1995;269:H72-9.

(14.) Rioufol G, Ovize M, Loufoua J, Pop C, Andre-Fouat X, Minaire Y. Ventricular fibrillation in preconditioned pig hearts: role of K+ATP channels. Am J Physiol. 1997;273:H2804-10.

(15.) Wu ZK, Iivainen T, Pehkonen E, Laurikka J, Tarkka MR. Ischemic preconditioning suppresses ventricular tachyarrhythmias after myocardial revascularization. Circulation. 2002;106(24):3091-6.

(16.) Rosen CL, Cote A, Haddad GG. Effect of enkephalins on cardiac output and regional blood flow in conscious dogs. Am J Physiol 1989; 256:H1651-1658.

(17.) Saxena P, Newman MA, Shehatha JS, Redington AN, Konstantinov IE. Remote Ischemic Conditioning: Evolution of the Concept, Mechanisms, and Clinical Application. J Card Surg. 2010 25:12-34.

Richard L. Summers, MD, Zizhuang Li, MD, PhD, Drew Hildebrandt, PhD

Department of Emergency Medicine, Department of Surgery, Division of Cardiothoracic Surgery Center for Excellence in Cardiovascular-Renal Research,University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216

Corresponding Author: Email: rsummers@umc.edu
Table 1. Comparison of the number of pigs with sustained ventricular
tachycardia and the average QT dispersion (QTdisp) and the average
percentage increase over control baseline values of QTdisp in control
and DADLE infused pigs during coronary occlusion. QTDisp was calculated
from 12 lead EKG records requiring a minimum of 6 leads. QTcAVG is the
average QT interval corrected for heart rate on these EKGs during the
occlusion period.

Group             No. With Sustained     Average No. of
                  Arrhythmia             EKG Leads

Dpigs (n = 6)     5 (83%)                6.8

Cpigs (n = 6)     1 (17%)                7.2

Group             QTcAVG                 Avg QTdisp
                  Msec (95%CI)           After Infusion

Dpigs (n = 6)     575(524,613)           144 msec

Cpigs (n = 6)     673(644,689)            61 msec

Group             % Increase
                  QTdisp

Dpigs (n = 6)     116 %

Cpigs (n = 6)      22 %

Table 2: Comparison of the hemodynamic effects of coronary occlusion
and infusion of DADLE vs control sham reported as percent of control
period average. Mean Pressure (mmHg of arterial); Heart Rate
(beats/min); Pulse Pressure (mmHg); DP/dt (mmHg/sec) are the averages
over their respective periods.

GROUP                  Mean Pressure          Heart Rate
                       (95%CI)                (95%CI)

Dpigs control          63                     104
                       (61,64)                (98,109)

Dpigs occlusion        63                     109
                       (58,68)                (106,112)

% of Control
Dpigs                  100 %                  104 %

Cpigs control          66                     86
                       (65,68)                (83,89)

Cpigs occlusion        59                     86
                       (58,60)                (85,87)

% of Control
cpigs                  89 %                   100 %

GROUP                  Pulse Pressure         DP/dt
                       (95%CI)                (95%CI)

Dpigs control          32                     716
                       (30,35)                (671,762)

Dpigs occlusion        31                     699
                       (30,32)                (679,720)

% of Control
Dpigs                  97 %                   98 %

Cpigs control          34                     523
                       (33,35)                (476,570)

Cpigs occlusion        27                     402
                       (26,28)                (385,420)
% of Control
cpigs                  79 %                   77 %
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Author:Summers, Richard L.; Li, Zizhuang; Hildebrandt, Drew
Publication:Journal of the Mississippi Academy of Sciences
Article Type:Report
Geographic Code:1U6MS
Date:Apr 1, 2011
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