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Ann Thorac Surg 2000;70:895-900
© 2000 The Society of Thoracic Surgeons

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Original articles: cardiovascular

Preconditioning of swine heart with monophosphoryl lipid A improves myocardial preservation

^a Department of Surgery, University of Connecticut School of Medicine, Farmington, Connecticut, USA
^b RIBI ImmunoChem Research, Inc, Hamilton, Montana, USA
^c Department of Surgery, Baystate Medical Center, Springfield, Massachusetts, USA

Address reprint requests to Dr Richard Engelman, Division of Cardiac Surgery, Baystate Medical Center, 759 Chestnut St, Suite 4628, Springfield, MA 01199

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. Ischemic preconditioning has been proven to be a powerful tool for myocardial protection in the setting of ischemia and reperfusion. A new drug to provide pharmacologic preconditioning, monophosphoryl lipid A (MLA), was administered 24 hours before an acute coronary occlusion in pigs to determine the effect on pharmacologic preconditioning.

Methods. Two studies were completed. In the first, swine were distributed into five groups: group I, control; group II,. aminoguanidine (AMG) (30 mg/kg), a selective inducible nitric oxide synthase (iNOS) blocker; group III, MLA (10 µg/kg); group IV, MLA (35 µg/kg); and group V, MLA and AMG (35 µg/kg and 30 mg/kg, respectively). Twenty-four hours after administration of the MLA, AMG, or both, regional left anterior descending coronary artery ischemia was induced for 15 minutes followed by one hour of global normothermic cardioplegic arrest and three hour reperfusion. Left ventricular function, tissue injury, and percentage of myocardial infarction were measured. Left ventricular myocardium in the left anterior descending coronary artery region was sampled for iNOS messenger RNA (mRNA) during ischemia and reperfusion. In the second study, pigs were sacrificed 0, 4, 6, 8, and 24 hrs after MLA/AMG administration for iNOS mRNA determination in nonischemic myocardium.

Results. Use of MLA significantly improved postischemic ventricular function, and reduced creatinine kinase release and percentage of infarction. Monophosphoryl lipid A induced expression of iNOS mRNA in nonischemic myocardium within four hours of administration which returned to base line by 24 hours. Normothermic regional ischemia then induced expression of iNOS mRNA, which returned to base line during reperfusion. Aminoguanidine completely abolished both MLA-induced and ischemia-induced iNOS mRNA and blocked the beneficial effects of MLA.

Conclusions. Use of MLA can provide myocardial preservation through enhanced expression of iNOS mRNA.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
The term ischemic preconditioning was introduced in 1986 by Murry, Jennings, and Reimer [1] to describe a phenomenon in which brief periods of myocardial ischemia followed by reperfusion improved myocardial tolerance to prolonged subsequent ischemia. It is now universally accepted that repeated ischemia and reperfusion can delay the onset of myocardial infarction [2], reduce postischemic ventricular dysfunction [3], and reduce the incidence of potentially fatal ventricular arrhythmias [4].

In clinical practice, ischemic preconditioning is not a practical approach for myocardial preservation during open heart surgery. Alternative approaches have been sought to mimic or duplicate ischemic preconditioning. These include heat shock [5], oxidative stress [6], pretreatment of hearts with adenosine [7] or its receptor agonist [8], the use of ATP-dependent potassium (K_ATP)channel openers [9], and, finally, exposure of the heart to hypoxic stress [10]. While most of these modalities are not clinically applicable, an ongoing quest for an acceptable pharmacological preconditioning stimulus has resulted in the discovery of a chemically modified nontoxic derivative of endotoxin, monophosphoryl lipid A (MLA), which has been found to render hearts more tolerant to ischemia/reperfusion injury [11]. Although MLA has been successful in preconditioning the hearts of a number of animal species—including the dog [12], rabbit [13], and rat [14 —it was not known whether MLA could precondition swine hearts, which in terms of coronary anatomy and collateral circulation are close counterparts to human hearts. Additionally, because our laboratory [14] has recently reported that MLA induces the synthesis and expression of iNOS in the rat heart exposed to ischemia, it was our goal to determine if a similar effect could be induced in the swine heart along with appropriate myocardial preservation.

	Material and methods

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Animal protocol
Animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (National Institutes of Health publication 85 to 23, revised 1985).

Two separate pig studies were performed. In the first, 48 swine were divided into five different groups: group I (n = 12) was a control group; group II (n = 6) received aminoguanidine (AMG), a selective inducible nitric oxide synthase (iNOS) blocker, at 30 mg/kg, subcutaneously, 24 hours before induction of ischemia; group III (n = 12) received monophosphoryl lipid A (MLA) 10 µg/kg administered intravenously (IV) 24 hours before the experiment; group IV (n = 12) received MLA 35 µg/kg IV administered 24 hours before the study; and Group V (n = 6) received of MLA 35 µg/kg IV plus AMG at 30 mg/kg, subcutaneously, both of which were injected 24 hours before the experiment. The schematic for this protocol is illustrated in Figure 1.

View larger version (25K): [in this window] [in a new window]   Fig 1. This schematic figure illustrates the experimental time line and the studies performed at various intervals; each group of subjects, described according to agents received, is denoted by an identifying symbol. Drugs were administered 24 hours before institution of cardiopulmonary bypass and regional ischemia and reperfusion. (AMG = aminoguanidine; CK = creatine kinase; CPB = cardiopulmonary bypass; INOS = inducible nitric oxide synthase; IV = intravenous; LAD = left anterior descending artery; LVF = left ventricular function; MLA = monophosphoryl lipid A; SQ = subcutaneous.)

Surgical procedure
At the time of the study, the pigs were tranquilized with intramuscular (IM) ketamine (20 mg/kg), (Bristol Meyers Squibb, Princeton, NJ) and anesthetized with sodium pentobarbital (25 mg/kg) (Abbot Laboratories, Abbott Park, IL). Endotracheal intubation was performed and ventilation maintained by a volume ventilator utilizing room air. A median sternotomy was performed, and the azygos vein ligated. After heparinization, an arterial cannula was placed in the ascending aorta through the right carotid artery and a venous cannula placed in the right atrium. A cannula was also placed in the left atrium through the appendage to control preload for ventricular function measurements. Cardiopulmonary bypass with a membrane oxygenator was initiated, and blood collected from the pig in a reservoir. The heart was then isolated in its own perfusion circuit by completely cross-clamping the ascending aorta just distal to the right brachiocephalic artery distal to the arterial inflow, and ligating both superior and inferior vena cavae. The main pulmonary trunk was drained. This achieves complete cessation of systemic circulation while coronary perfusion is maintained.

Once stable bypass was established, the left anterior descending coronary artery (LAD) was snared just distal to the first diagonal branch. After l5 minutes of normothermic regional ischemia, the aorta was clamped and the ligature removed from the LAD. Normothermic cardioplegic arrest was initiated. Initial high-potassium blood cardioplegic solution (K⁺ 20 to 24 mEq/l, 20 ml/kg) was administered through the carotid cannula into the coronary circulation, administered at a flow rate to insure a perfusion pressure of 50 to 75 mm Hg. This high-potassium blood cardioplegic solution arrested each heart promptly. Additional low-potassium blood cardioplegic solution (K⁺, 8 to 12 mEq/l, 10 ml/kg) was administered every 15 minutes (at 15, 30, and 45 minutes) for a total of 60 minutes of arrest.

After 60 minutes of cardioplegic arrest, the heart was reperfused on cardiopulmonary bypass. Normothermia was maintained with a heat exchanger, and reperfusion was continued for a total of 180 minutes. Defibrillation was applied when the heart suffered ventricular fibrillation during reperfusion. The heart was atrially paced at 120 beats/min. No cardiotonic or antiarrhythmic drugs were administered during the experiment. Blood samples were taken through the pulmonary arterial cannula (retrieving coronary sinus blood) at 0 (baseline), 60, 120, and 180 minutes of reperfusion for creatine kinase (CK) measurement. Left ventricular myocardial needle biopsies were taken from the LAD region before regional ischemia, during cardioplegic arrest, at 30 and 60 minutes, and after one, two, and three hours of reperfusion to extract mRNA for determination of iNOS expression in groups IV (MLA) and V (MLA + AMG).

Measurement of myocardial function
Functional data were obtained by adding 60 ml of saline through the left atrial cannula to raise the left ventricular end-diastolic pressure and subsequently withdrawing the saline while measuring maximal developed pressure and end-diastolic pressure before ischemia (control value) and at 30, 60, 90, 120 and 180 minutes of reperfusion.

Measurement of CK release
Creatine kinase was quantified from 0.5 ml of plasma obtained before LAD occlusion and for samples withdrawn every 60 minutes during reperfusion. The enzymatic assay method was employed, using a CK assay kit (Sigma Diagnostics, St. Louis, MO). The absorbance was read at 340 nanometers using a Beckman DU-8 spectrophotometer (Beckman Coulter, Fullerton, CA).

Infarct size measurement
After three-hour reperfusion and completion of all studies, triphenyl tetrazolium chloride (10 ml, 1% solution) in phosphate buffer preheated to 37°C was injected directly into the LAD. With this solution, noninfarcted myocardium is stained red and infarct remains unstained after a 10-minute incubation period,. The heart was stored at -20°C, and frozen slices were analyzed with sections from apex to base. The infarct area as a percent of total left ventricular (LV) myocardium was calculated.

Thin sections were placed between plates and panned using National Institutes of Health Image processing software. Each digitized image was subjected to background subtraction and contrast enhancement. The total LV area and the area of infarction were traced, and the respective areas calculated in terms of pixels. The infarct volume was calculated and the sum of all slices used to compute a percent infarct/total LV area.

Determination of iNOS gene expression by Northern blot analysis of iNOS and mRNA
Biopsies were obtained for iNOS and mRNA determination in the 30 pigs sacrificed at 0, 4, 6, 8, and 24 hours following MLA ± AMG pretreatment, as well as from the biopsies obtained from the LAD (ischemic) regions in groups IV and V before ischemia; at 30 and 60 minutes of cardioplegic arrest; and at 1, 2 and 3 hours of reperfusion. Biopsies were immediately frozen in liquid nitrogen and stored at -70°C for subsequent mRNA determination. Total RNA was extracted from the heart in our laboratory by the acid-guanidinium-thiocyanate-phenol-chloroform method as previously described [15]. For Northern blot analysis, total RNA underwent electrophoresis in 1% agarose-formaldehyde-formamide gel and was transferred to a Genescreen Plus membrane (NEN Life Science Products, Boston, MA). After prehybridization, membranes were then hybridized with a 1.8-kb fragment of mouse macrophage iNOS; copy DNA (cDNA) was obtained from Cayman Chemical Company (Ann Arbor, MI). Each hybridization was repeated at least three times using different membranes. After each hybridization, the iNOS cDNA was removed and rehybridized with a glycerol-3-phosphate dehydrogenase (GPDH) cDNA probe, the results of which served as loading controls. The autoradiograms were quantitatively evaluated by a computerized ß scanner (Model #860-PC, Molecular Dynamics, Sunnyvale, CA). The results of densitometric scanning were normalized relative to the signal obtained with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe.

Statistical analysis
All data were expressed as mean ± SEM. Data were analyzed by a two-way analysis of variance for repeated measured followed by a multiple-comparison Scheffé test to determine differences between groups. Significance was considered to be present at a p value of less than or equal to 0.05.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Myocardial function
Hemodynamic measurements under an intraventricular load of 60 ml of saline in control and treatment groups are shown in Figure 2. As evident in the figure, left ventricular developed pressure did not vary between groups during baseline measurements, while MLA at 35 µg/kg had the best recovery of left ventricular systolic function. Monophosphoryl lipid A at 10 µg/kg also showed some improvement, but the differences were significant only at two hours, as compared to the corresponding control group. Aminoguanidine blocked the beneficial effects of MLA but by itself had no effect on myocardial function; the results were identical with vehicle control. The left ventricular end-diastolic pressure rose significantly in all groups during reperfusion, but did not differ between them at any time point. The first-time derivative of left ventricular pressure (dP/dt) was also measured; it duplicated the results shown for left ventricular developed pressure.

View larger version (46K): [in this window] [in a new window]   Fig 2. Illustration of left ventricular developed pressure. *p<0.05 versus control; p<0.05 versus MLA 35 µ/kg alone.

View larger version (17K): [in this window] [in a new window]   Fig 3. Creatine kinase release during reperfusion for the five groups *p<0.05 versus control (Group I = [open square]; group II = [black triangle]; group III = [black square]; group IV = [black diamond]; group V = [black circle].)

View larger version (66K): [in this window] [in a new window]   Fig 4. Infarct size in each of the five groups. *p < 0.05 for decrease in infarct size versus control in MLA groups. p less than 0.01 versus MLA alone (AMG = aminoguanidine; MLA = monophosphoryl lipid A.)

View larger version (31K): [in this window] [in a new window]   Fig 5. iNOS generation at intervals after MLA and MLA + AMG administration. *p < 0.05 increase in iNOS generation versus MLA + AMG (AMG = aminoguanidine; MLA = monophosphoryl lipid A.)

View larger version (34K): [in this window] [in a new window]   Fig 6. iNOS generation during ischemia and reperfusion in the ischemic zone. A significant increase (p < 0.01) in iNOS generation was observed in the 35-µg/kg MLA group during ischemia but not in the MLA + AMG group.(AMG = aminoguanidine; iNOS = inducible nitric oxide synthase; MLA = monophosphoryl lipid A.)

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

Mammalian hearts can be adapted to tolerate ischemia by subjecting them to a small, therapeutic amount of stress; this process of adaptation has been termed "preconditioning." The phenomenon was originally referred to as "ischemic preconditioning," and was achieved by repeated brief episodes of ischemia and reperfusion [1]. Ischemic preconditioning was originally described as a process that delays or slows ischemic cell death. It is now universally accepted that a small amount of stress associated with repeated ischemia and reperfusion can not only delay the onset of further irreversible injury (infarction) but can also reduce postischemic reversible ventricular dysfunction (stunning) and the incidence of reperfusion arrhythmias [1–10]. Preconditioning may be one of the most powerful tools to achieve myocardial protection against ischemia and reperfusion.

Preconditioning can be induced by a number of different approaches, including stimulation of a variety of receptors (eg, adenosine-A1 [8], bradykinin [16], and -1-adrenoceptor [17]), opening of K_ATP channels [9], inducing heat shock [5], oxidative stress [6], and hypoxia [10].

Despite a great deal of research into the preconditioning phenomenon over the last decade, the pathophysiology and the mechanisms by which preconditioning exerts cardioprotection remain controversial. It is generally believed that one or more intracellular mediators rapidly released in response to stress is responsible for preconditioning. Although there is controversy regarding the pathway for signal transduction, it is generally accepted that protein kinase C is involved in signaling this adaptive response [18].

Ischemic preconditioning is not easily applicable in the clinical arena. During open heart surgery it is impractical to precondition a heart by subjecting it to repeated ischemia and reperfusion, a measure that is, in fact, of questionable efficacy. To resolve this issue, several pharmacologic compounds have been developed that can mimic preconditioning. Recently, 24-hour pretreatment of rabbit heart with MLA, a chemically modified nontoxic derivative of endotoxin, was found to render hearts more tolerant to ischemia/reperfusion injury [11]. Monophosphoryl lipid A is derivatized from the minimal pharmacore of endotoxin (lipid A) by removing a phosphodiester from the reducing glucosamine sugar of the disaccharide followed by saponification of a long chain ß-hydroxy ester from the three-position hydroxyl group of the reducing glucosamine. Pretreatment 12 to 24 hours before ischemia with a single intravenous bolus injection of MLA was found to cause 50% to 75% reduction of infarct size in canine and rabbit hearts [12, 13]. Adenosine triphosphate–sensitive potassium channels have been shown to play a role in MLA preconditioning of hearts, because K_ATP channel blockers were shown to abolish the beneficial effects of such preconditioning [19]. Additionally, MLA has been found to protect the heart from ischemic reperfusion injury manifested as infarction, arrhythmias, or stunning [20]. Because MLA can exert its cardioprotective effect 24 hours after treatment, protection may be mediated by transcriptional regulation of one or more genes and their translation into effector proteins. Indeed, transcription of iNOS in rats [14] and iNOS protein activation in rabbits [11] have been found to be involved in MLA-mediated cardioprotection.

Preconditioning is known to be a species-specific phenomenon; different mechanism are found in rats, rabbits, pigs, and dogs. For example, adenosine A1 receptor agonists can precondition rabbit hearts, but these receptors have no preconditioning effect on rat hearts [21]. Inhibition of protein kinase C can block the effects of ischemic preconditioning in both rat and rabbit myocardium but is quite ineffective in dog hearts [22]. The results of this study demonstrate that pig hearts can be pharmacologically protected from ischemia and reperfusion injury by MLA in animals treated with the drug 24 hours before cardiopulmonary bypass. Monophosphoryl lipid A exerts a cardioprotective effect in this model, as evidenced by the reduction of myocardial infarction and CK release and by improved function during reperfusion.

It is apparent in this study that MLA induces iNOS generation as early as 4 hours after administration, with iNOS generation reaching a peak at 8 hours and declining to baseline by 24 hours. We also documented enhanced gene expression 24 hours after MLA administration induced by LAD ischemia. In fact, this second window of preconditioning occurred in this study after 15 minutes of ischemia. This duplicates the work of Zhao and associates [11], who found iNOS stimulation after MLA pretreatment only in ischemic rabbit myocardium at 15 and 30 minutes; iNOS stimulation was absent in nonischemic tissue. These investigators [11] suggested that MLA-induced iNOS protein was present in the rabbit heart in an inactive form requiring activation by ischemia. They proposed that ischemia would activate kinases or inhibit phosphatases which could then promote phosphorylation of the inactive iNOS initially generated by MLA. The generation of iNOS in turn would stimulate nitric oxide (NO) production.

Nitric oxide is an important agent in cardioprotection [23, 24]. A recent study from our laboratory has demonstrated that NO enhances myocardial protection by a cyclic guanosine monophospate (cGMP)–dependent [25] as well as a cGMP-independent mechanism [23]. Nitric oxide is a unique messenger in that it is produced in one cell and diffuses into adjacent target cells to activate cytosolic guanylate cyclase–bound heme to generate the NO-heme adduct of guanylate cyclase [23–25]. In the ischemic myocardium, NO also functions by a cGMP-independent mechanism, because of its antioxidant effects towards both oxygen-free radicals and oxyferryl myoglobin radicals, which were important causative factors for ischemia reperfusion injury [23–24]. It has generally been accepted that one pathway for NO affords cardioprotection by its ability to quench free radicals generated during reperfusion of ischemic myocardium. Thus, NO seems to serve both as an intracellular antioxidant and as a cardioprotective messenger molecule in the ischemic myocardium.

The results of this study indicate that pig hearts, similar to rat, rabbit, and dog hearts, can be pharmacologically adapted to ischemia by MLA. Such adaptation in this study provides beneficial effects in two ways, by reducing myocardial infarction and tissue injury and by improving cardiac function. The adaptation process appears to involve induction of the expression of the iNOS gene, its activation during ischemia, and its inhibition by the iNOS selective enzyme inhibitor AMG.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
This study was supported by NIH HL22559, HL 34360 and a Grant-in-Aid from the American Heart Association, as well as by a grant from RIBI ImmunoChem Research.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Richard M. Engelman Daniel T. Engelman Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yoshida, T. Articles by Das, D. K. Search for Related Content PubMed PubMed Citation Articles by Yoshida, T. Articles by Das, D. K.