A single brief period of global myocardial ischemia produced by ventricular overdrive pacing (VOP) renders the heart resistant to electrophysiologic and hemodynamic consequences of a second VOP-induced ischemic episode (1). This type of protection, often referred to as early or "classic" preconditioning proved to be of short duration, lasting for ∼60 min (1). Nevertheless, when preconditioning was induced by a series of VOP periods, not only the short-term but a long-term protection, often referred to as the "second window of protection" or SWOP, was manifested. The duration of this latter exceeded 24 h in rabbits (2). Moreover, the short-term protection by a single period of VOP is suggested to be a healthy heart phenomenon because it was found blocked in diseased states such as hypercholesterolemia/atherosclerosis (3); the long-term protection, however, afforded by multiple preconditioning VOP periods was preserved even in the atherosclerotic state (4). The preconditioning phenomenon involved in either form of protection is attributed to an increased formation/release of endogenous protective substances such as adenosine, prostacyclin, and nitric oxide (NO), which are currently subjects of possible pharmacologic exploitation (5).
One potentially promising therapeutic approach to elicit cardioprotection in clinical instances of planned myocardial ischemia is the application of the endotoxin derivative monophosphoryl lipid A (MLA; 6). Pretreatment with MLA 24 h before a planned ischemic insult was found to preserve left ventricular function, attenuate adenosine triphosphate (ATP) breakdown, decrease creatine kinase release, and reduce infarct size in hearts of rabbits, rats, and dogs subjected to myocardial ischemia/reperfusion (7-11). Therefore Przyklenk et al. (11) suggested that MLA is a "SWOP mimetic" in a canine model of cardiac ischemia. Because SWOP seems to work in atherosclerosis (4), our study was concerned with the possibility that pretreatment with MLA, a recently developed nontoxic analog of endotoxin, confers some protection on the ischemic heart of atherosclerotic conscious rabbits. We used electrophysiologic and hemodynamic changes produced by a single period of VOP to test the possible delayed antiischemic effect of pretreatment with a single intravenous dose of MLA in conscious rabbits.
MATERIALS AND METHODS
Animals and surgery
The experiments were carried out with 40 adult, male New Zealand White rabbits, weighing 3.0-3.5 kg, housed as described (1,12). The animals were fed commercial laboratory chow and tap water ad libitum. Surgery was performed under aseptic conditions after a 2-week adaptation period. The rabbits were anesthetized with an intravenous bolus of diazepam (Sigma, St Louis, MO, U.S.A.; 10 mg/kg body weight) and ketamine (EGIS Pharmaceuticals, Budapest, Hungary; 5 mg/kg); lidocaine (EGIS Pharmaceuticals; 10 mg/kg) was given for local pain relief. A bipolar 4F electrode catheter (Cordis, Erkrath, Germany) was introduced into the right ventricle through the main branch of the right jugular vein to obtain the intracavitary electrogram. The correct position of the catheter was confirmed by analyzing the intracavitary electrogram as described (12). Almost the whole catheter was fixed under the skin of the neck, making a subcutaneous cradle for the precurved device. The proximal part of 4 mm length of the electrode was exteriorized through a separate incision. During the experiments, this part of the catheter was fitted to a two-channel programmable stimulator (Experimetria, London, U.K.) through a special connector for 4F catheters (Cordis). A polyethylene cannula was inserted into the left ventricular cavity through the right external carotid artery to measure left ventricular pressure. The proximal part of the cannula was exteriorized through a separate incision and connected to an Experimetria UK two-channel electromanometer through a Statham P23 Db Transducer (Gould, Balainviliers, France); another polyethylene cannula was introduced into the distal third of the central ear artery for recording mean arterial blood pressure (MABP). This latter cannula was connected to the second channel of the electromanometer through a separate Statham transducer. The volumes of both cannulas were precisely determined before surgery, and each cannula was then filled with appropriate volume of Na-heparin (5,000 IU/ml; Richter Pharmaceuticals, Budapest, Hungary) to prevent blood coagulation in the cannulas. Because of the soft surgery technique, no systemic antibiotic treatment was necessary; nevertheless, 106 IU penicillin (Zyma-Biogal Pharmaceuticals, Debrecen, Hungary) was applied locally before closing the neck wound. A 7-day period of convalescence was allowed for each animal before commencement of the experiments. During the first 24 h after surgery, mean body temperature transiently increased by 0.8 ± 0.16°C, and a 10-15% weight loss occurred. One week later, the body weight had returned to the presurgery value; weight gain did not exceed 10% of the baseline during the experiments. The rectal temperature remained normal throughout the experiments, irrespective of any treatment schedules.
Cardiac electrophysiology and hemodynamics
The right intracavitary electrogram, the chest-lead ECG, the left ventricular pressure curve and MABP were continuously recorded by means of an Experimetria UK 6-channel multiscriptor. Right ventricular effective refractory period (VERP) was determined as described (1,12). In brief, electrical square impulses of 1.5-ms duration at twice diastolic threshold were delivered through the implanted electrode by means of the programmable stimulator. Single programmed extrastimuli were introduced late in diastole after 15 basic driven beats of 200-ms cycle length. The coupling intervals of the programmed stimuli were then gradually shortened in 2-ms steps until disappearance of signs of ventricular activation in the chest-lead ECG. The longest coupling interval that failed to produce QRS complexes in the chest-lead ECG was referred to as VERP.
Global myocardial ischemia induced by ventricular overdrive pacing
The induction of global myocardial ischemia was based on a method described elsewhere (1). Conscious rabbits were subjected to VOP at 500 beats/min with double-threshold square impulses of 1.5-ms duration over a 10-min period. The resulting (postpacing) right intracavitary ST-segment elevation, increase in left ventricular end-diastolic pressure (LVEDP), and shortening of VERP were measured to characterize the severity of ischemia. Postpacing ST-segment elevation was determined 40 ms after the "J" points. LVEDP was determined after the "a" (atrial activation) wave immediately before the first phase of LVP upstroke, appearing simultaneous with the appearance of the "s" wave in the chest-lead ECG. The s wave in the chest-lead ECG was used as a reference point for LVEDP determination to assure that the same point in the cardiac cycle was examined in each case. Mean values of five consecutive cardiac cycles before and after VOPs produced the data for evaluation. VERP was measured under resting conditions (before VOP) and 20-30 s after cessation of VOP (postpacing values).
At day 7 of convalescence after surgery, the animals were randomly divided into two groups. Thirty animals continued to feed on ordinary laboratory chow (normal group), whereas the other group of 30 rabbits was started on laboratory chow enriched with 1.5% cholesterol (atherosclerotic group) over a period of 8 weeks. According to our experiences, exposure to a cholesterol-enriched diet over this period results in a nearly 20-fold increase in serum total cholesterol level with aortic lesion surface area of 55% and an ∼50% loss of endothelium-dependent relaxation of rings from thoracic aortae in rabbits (3,4).
The animals in each group were randomly divided into three treatment subgroups (10 in each). The first subgroup of rabbits received the solvent for MLA, the second group was treated with 10 μg/kg MLA, whereas the third subgroup of animals received 30 μg/kg MLA.
The animals in each group underwent the experimental protocol as follows.
The rabbits were subjected to VOP (500 beats/min over a 10-min period), and both pre- and postpacing electrophysiologic and hemodynamic variables were determined (Monday). After a 3-day interval, various single intravenous doses of MLA or its solvent or both were administered (Thursday). The animals were subjected to a second VOP of the same rate and duration as the first, 24 h after administration of the substance/its solvent, and pre- and posttreatment values of the parameters measured were determined again (Friday). Schematic representation of the experimental protocol used in the atherosclerotic and normal groups is seen in Fig. 1. The rationale for the use of electrophysiologic and hemodynamic changes produced by a single period of VOP to study antiischemic drug effects was discussed in detail (1-4,12). These changes are highly reproducible, and their severity is fairly proportional to the rate or duration or both of VOP, thus raising the possibility of making comparisons among several antiischemic therapeutic maneuvers and drug effects. In "self-control" experiments, however, attention is paid to the VOP-induced preconditioning phenomenon (i.e., a single VOP period is able to confer short-term protection on the rabbit heart in situ from consequences of a subsequent VOP period). The duration of this short-term protection, however, does not exceed 2 h and no SWOP phenomenon was seen in rabbits after single VOP periods (1-4). Therefore the 3-day interpacing interval left between the control (first VOP) and the subsequent test VOPs (second VOP) excludes the influence of the first VOP period on either the effect of MLA or the second pacing.
Eighteen rabbits had to be excluded from the study, so that seven rabbits were included in each experimental subgroup with total number of 42 animals of the whole study. The causes of exclusions included postsurgery pulmonary embolism (in four rabbits); mechanical damage to the implanted electrodes (in 11 cases); one rabbit in the atherosclerotic group with cholestatic jaundice due to gallstones (the animals in the experimental groups did not reveal any significant increase in serum bilirubin level, and other liver-function tests were also normal throughout the study); one with severe ventricular arrhythmias in response to VOP; and one animal in which VOP failed to produce intracavitary ST-segment elevation.
All data are expressed as mean ± standard deviation (SD). Electrophysiologic and hemodynamic parameters were statistically analyzed by analysis of variance (ANOVA) followed by a modified t test for unpaired data. The p values were adjusted according to Bonferroni's method (13). Changes were considered statistically significant at p < 0.05.
Myocardial ischemia in normal animals
Hemodynamic parameters. Heart rate was not modified by VOP (i.e., postpacing heart rate was the same as that measured before pacing; Table 1), whereas postpacing MABP was significantly lower than corresponding prepacing values (Table 2). VOP significantly increased LVEDP (Fig. 3).
Electrophysiologic alterations. VOP induced a highly reproducible intracavitary ST-segment elevation (Fig. 2) and a significant reduction of VERP (Fig. 4).
Myocardial ischemia in atherosclerotic animals
Hemodynamic parameters. VOP was without effect on heart rate in the atherosclerotic animals (Table 1). Postpacing decrease in MABP and LVEDP increase, however, were more pronounced than in the normal rabbits (Table 2 and Fig. 3). Resting values for both MABP (Table 1) and LVEDP (Fig. 3) were higher in the atherosclerotic group than the corresponding values in the normal group.
Electrophysiologic parameters. Intracavitary ST-segment elevation was significantly higher in the atherosclerotic group than in the normal one (Fig. 2), whereas VOP-induced VERP reduction was approximately the same in the two groups (Fig. 4).
Effect of MLA on resting electrophysiologic and hemodynamic parameters in conscious rabbits
MLA or its solvent or both were without effect on resting heart rate, LVEDP, or MABP in normal animals. Ventricular refractoriness, however, was significantly increased by 30 μg/kg MLA in both normal and atherosclerotic rabbits (Fig. 4,) and pretreatment with 30 μg/kg MLA decreased MABP and LVEDP in the atherosclerotic animals (Table 2 and Fig. 3). The solvent for MLA was without effect on any of the resting parameters measured.
Effect of MLA on VOP-induced hemodynamic changes in normal animals
Pretreatment with both 10 and 30 μg/kg MLA abolished the marginal decrease in MABP in response to VOP (Table 2), and 30 μg/kg MLA significantly decreased postpacing heart rate. VOP-induced LVEDP-increase was significantly attenuated by either dose of MLA (Fig. 3). The solvent for MLA was without effect.
Effect of MLA on VOP-induced electrophysiologic changes in normal animals
MLA decreased VOP-induced reduction of VERP at the dose of 30 μg/kg. The lower MLA dose was without effect (Fig. 4). Postp acing intracavitary ST-segment elevation was significantly attenuated by both MLA doses. The higher dose, however, produced a more marked antiischemic effect (Fig. 2).
Effect of MLA on VOP-induced hemodynamic changes in atherosclerotic rabbits
The decrease in MABP by VOP was completely abolished by the MLA doses studied (Table 2), and the endotoxin derivative significantly decreased postpacing heart rate at either dose applied (Table 1). The postpacing LVEDP increase was significantly moderated by pretreatment with either dose of MLA (Fig. 3). It should be noted again, however, that the higher MLA dose was able to restore LVEDP in the resting state in the atherosclerotic animals.
Effect of MLA on VOP-induced electrophysiologic changes
Intracavitary ST-segment elevation was significantly decreased by the MLA doses studied. The protection by 30 μg/kg MLA exceeded that produced by the lower dose (Fig. 2). Postpacing VERP reduction also was reduced by MLA, so that no VERP shortening was seen in animals pretreated with 30 μg/kg MLA (Fig. 4).
The results presented here confirm previous findings that a 24-h pretreatment with a single intravenous bolus of MLA attenuates hemodynamic changes resulting from a planned ischemic episode (6,9). It is also shown that intracavitary ST-segment elevation, an indicator of myocardial ischemia in our model, and VOP-induced reduction of ventricular refractoriness (1) are also significantly diminished by MLA pretreatment in conscious rabbits. However, the major original observation of our work is that MLA is able to elicit these late protective effects even in hypercholesterolemic/atherosclerotic conscious animals.
Previous studies demonstrated a similarity between protection conferred by MLA and the antiischemic effect characteristic for the so-called SWOP, the delayed phase of preconditioning. This similarity consists of a decreased neutrophil infiltration into ischemic tissue (8), preservation of high-energy phosphates (10), and an enhanced 5′-nucleotidase activity on reperfusion (11). These changes are characteristic of both preconditioning (14-16) and the late-appearing protective effect of endotoxin analogs (6). Although classically, ischemic preconditioning is studied in the context of myocardial necrosis resulting from regional ischemia produced by coronary artery occlusion, it is becoming apparent that preconditioning offers wide-ranging protection against other undesirable consequences of myocardial ischemia, such as depression of contractile function and arrhythmia precipitation. MLA and lipopolysaccharide (LPS) have also been shown both to reduce infarct size and to improve postischemic cardiac function (17,18).
According to findings of some very recent studies, activation of cardiac ATP-sensitive potassium (KATP) channels (known to be of importance in cardioprotection by preconditioning) might be relevant in the antiischemic effect of endotoxin analogs (6). Opening of KATP is proposed to confer protection by reducing the action-potential duration, which translates to less time in contraction; both spare ATP and reduce calcium entry, a putative mediator of ischemic injury. Nevertheless, our results do not seem to confirm the exclusive role of the KATP-opening mechanism in the antiischemic effect of MLA because VERP reduction by global ischemia was inhibited in the MLA-treated animals, an opposite effect that would result from KATP opening. In a recent study we found that cromakalim, a prototype of KATP openers, enhanced ischemic VERP shortening in parallel with a marked anti-ischemic effect (19), whereas when combined with cicletanine, a specific cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) inhibitor, no VERP reduction was seen without impairment of the antiischemic potency compared with that produced by either drug, separately. Therefore KATP channel activation alone does not seem to mimic the antiischemic effects of MLA in conscious rabbits. There is substantial pharmacologic evidence that the guanylate cyclase-cGMP-PDE system may contribute to cardioprotection produced by preconditioning (1,3,5,19,20). Thus it is suggested that when KATP-channel opening appears in relation to or in parallel with activation of the cardiac cGMP system, a marked antiischemic effect is seen with no reduction of ventricular refractoriness. Because the cardioprotective effect of MLA has been shown to be underlain by an enhancement in activity of the cardiac inducible NO synthase (18), the MLA-induced antiischemic effect resembles that produced by either preconditioning (4,21) or a combined administration of cromakalim and a cGMP-increasing agent (19). We think that the ability of MLA to alleviate VERP reduction in the ischemic tissue might serve as an explanation for the antiarrhythmic effect of the endotoxin derivative seen in other models of myocardial ischemia/reperfusion (22). Although our work was not conducted to study the overall protective mechanism of action of MLA in hypercholesterolemia and atherosclerosis, an enhancement in cardiac NO by MLA (18) might underlie the beneficial effect of this substance in our model, which is characterized by a deficiency in NO synthesis/effect (3,4,23). Indeed, ischemic changes produced by VOP of standardized rate and duration were significantly aggravated by the 8-week exposure to a cholesterol-rich diet, a finding consistent with that of Hoshida et al. (24), who described that infarct size produced by a 30-min coronary artery occlusion followed by 2-h reperfusion was significantly augmented by hypercholesterolemia in rabbits maintained on 1% cholesterol-rich diet over a 10-week period. They also found that N-nitroso-N-acetylpenicillamine, an exogenous NO donor, inhibited the reduction in the ischemic tolerance of the atherosclerotic heart. Speculatively, an enhancement of endogenous NO production by MLA might yield the same consequence.
In summary, the results show that 24-h pretreatment with MLA produces an antiischemic effect in conscious rabbits with myocardial ischemia induced by rapid ventricular pacing. This protective effect is characterized by an attenuation of VOP-induced increase in LVEDP, intracavitary ST-segment elevation, and ischemia-induced reduction of ventricular refractoriness. Moreover, 30 μg/kg MLA was found to produce a moderate prolongation of resting VERP, suggesting that pretreatment with MLA results in an antiarrhythmic effect, enhancing its own possible protective effect on ventricular arrhythmias resulting from myocardial ischemia. The results also indicate that the protective mechanisms induced by MLA pretreatment are functional in experimental hypercholesterolemia and atherosclerosis. In addition to its cardioprotective effect, the ability of MLA to restore blood pressure in hypercholesterolemic/atherosclerotic animals may accentuate the possible benefit of this substance to confer protection on the ischemic heart in diseased states associated with hypercholesterolemia and atherosclerosis.
1. Szilvassy Z, Ferdinandy P, Bor P, Jakab I, Lonovics J, Koltai M. Ventricular overdrive pacing-induced anti-ischemic effect: a conscious rabbit model of preconditioning. Am J Physiol
2. Szekeres L, Papp JGy, Szilvássy Z, Udvary É, Végh Å. Moderate stress by cardiac pacing may induce both short term and long term cardioprotection. Cardiovasc Res
3. Szilvassy Z, Ferdinandy P, Szilvassy J, et al. Loss of preconditioning in atherosclerotic rabbits: the role of hypercholesterolaemia. J Mol Cell Cardiol
4. Szekeres L, Szilvassy Z, Ferdinandy P, Nagy I, Karcsu S, Csati S. Delayed cardiac protection against harmful consequences of stress can be induced in experimental atherosclerosis in rabbits. J Mol Cell Cardiol
5. Parratt JR. Possibilities for the pharmacological exploitation of ischaemic preconditioning. J Mol Cell Cardiol
6. Elliott GT. Pharmacologic myocardial preconditioning with monophosphoryl lipid A (MLA) reduces infarct size and stunning in dogs and rabbits. Ann N Y Acad Sci
7. Nelson DW, Brown JM, Banerjee A, et al. Pretreatment with a nontoxic derivative of endotoxin induces functional protection against cardiac ischaemia/reperfusion injury. Surgery
8. Yao Z, Auchampach JA, Pieper GM, Gross GJ. Cardioprotective effects of monophosphoryl lipid A, a novel endotoxin analogue, in the dog. Cardiovasc Res
9. Elliott GT, Zhao L, McLeod RA, Floyd KW, Graves GM. Cardiac functional preservation by monophosphoryl lipid A in a rabbit model of prolonged regional cardiac ischemia and reperfusion. Int J Immunother
10. Zhao L, Kirsch C, Hagen SR, Elliott GT. Preservation of global cardiac function in the rabbit following protracted ischaemia/reperfusion using monophosphoryl lipid A (MLA). J Mol Cell Cardiol
11. Przyklenk K, Zhao L, Kloner R, Elliott GT. Cardioprotection with ischemic preconditioning and MLA: role of adenosine-regulating enzymes? Am J Physiol
12. Szilvássy Z, Jakab I, Bor P, et al. Anti-ischemic effect of cicletanine in conscious rabbits: a possible relation to changes of cardiac cyclic nucleotide content. Coron Artery Dis
13. Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res
14. Murry CE, Richard VJ, Reimer KA, Jennings RB. Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode. Circ Res
15. Kitazake M, Hori M, Takashima S, Sato H, Inoue M, Kamada T. Ischemic preconditioning increases adenosine release and 5′-nucleotidase activity during myocardial ischemia and reperfusion in dogs. Circulation
16. Mizumura T, Nithipatikom K, Gross GJ. Bimakalim, an ATP-sensitive potassium channel opener mimics the effect of preconditioning to reduce infarct size, adenosine release and neutrophil function in dogs. Circulation
17. Rowland RT, Cleveland JC, Meng X, Ho L, Harken AH, Brown JM. A single endotoxin challenge induces delayed myocardial protection against infarction. J Surg Res
18. Zhao L, Weber P, Smith JR, Comerfors ML, Elliott GT. Role of inducible nitric oxide synthase in pharmacological "preconditioning" with monophosphoryl lipid A. J Mol Cell Cardiol
19. Szilvássy Z, Koltai M, Ferdinandy P, et al. Cromakalim and cicletanine against pacing-induced myocardial ischemia in conscious rabbits. Life Sci
20. Ferdinandy P, Szilvássy Z, Balogh N, et al. Nitric oxide is involved in active preconditioning in isolated working rat hearts. Ann N Y Acad Sci
21. Bolli R, Bhatti ZA, Tang X-L, et al. Evidence that late preconditioning against myocardial stunning in conscious rabbits is triggered by the generation of nitric oxide. Circ Res
22. Vegh A, Papp JG, Parratt JR. Pretreatment with monophosphoryl lipid C (MPL-C) reduces ischemia-reperfusion-induced arrhythmias in dogs [Abstract]. J Mol Cell Cardiol
23. Szilvassy Z, Nagy I, Szilvassy J, Jakab I, Csati S, Lonovics J. Impaired nitrergic relaxation of the sphincter of Oddi of hyperlipidaemic rabbits. Eur J Pharmacol
24. Hoshida S, Nishida M, Yamashita N, et al. Amelioration of severity of myocardial injury by a nitric oxide donor in rabbits fed a cholesterol-rich diet. J Am Coll Cardiol