4′-Monophosphoryl lipid A (MLA) is an attenuated derivative of lipopolysaccharide that lacks many of the endotoxic properties of the parent molecule but retains potent adjuvant and immunostimulating activities (1-4). MLA has been shown to be safe and well tolerated in humans at doses at which it is still a potent immunostimulant (5) and attenuates the systemic response to bacterial endotoxin (6). Recent studies showed that MLA is cardioprotective when administered 24 h before sustained ischemia and reperfusion (I/R) in rats (7,8), rabbits (9-11), and dogs (12). Although the mechanism by which MLA protects the myocardium is still undetermined, opening of the adenosine triphosphate (ATP)-sensitive potassium channel (KATP channel) has been implicated because glibenclamide, a sulfonylurea KATP channel antagonist, abolished the cardioprotective effect of MLA (13). Although glibenclamide is a relatively selective blocker of KATP channel (14), its binding is inhibited by Mg2+-ATP (15), and it may be a competitive receptor antagonist of the KATP channel activators (16). Thus, to further test whether KATP channel is involved in MLA-induced pharmacologic preconditioning (PC), we chose to use a more selective KATP channel blocker, 5-hydroxydecanoate (5-HD). 5-HD is a proposed class III antiarrhythmic agent (14), a natural lipid component of human milk (17). It is structurally distinct from sulfonylureas (17) and has no systemic metabolic effects (14). 5-HD does not depress the inward rectifier K+ channel but selectively inhibits the KATP channel (17). The blocking action of 5-HD is comparable to that of ATP, with a stoichiometry for binding on the channel approaching 1:1 and a blockade of the channel without changing the gating of the channel during bursts (17). 5-HD does not inhibit channel activity by modulating the binding of ATP and may act on the same binding sites on the channel as does ATP (17). Furthermore, the ability of 5-HD to reverse the postischemic improvement in cardiac function of KATP channel activators without affecting their preischemic actions suggests that 5-HD is ischemia selective (14). The intracoronary infusion of 5-HD has been shown to abolish the myocardial protective effects of ischemic PC in barbitone-anesthetized dogs, by Auchampach et al. (18), independent of differences in hemodynamics, coronary collateral blood flow, or size of the ischemic bed.
All procedures involving animals were conducted in conformity with the guidelines of the Committee on Animals of Virginia Commonwealth University and the National Institutes of Health (NIH) "Guide for the Care and Use of Laboratory Animals" [DHHS Publication No. (NIH) 80-23, Revised, Office of Science and Health Reports, Bethesda, MD 20205].
Animals were randomly divided into four groups.
- Group I (MLA-vehicle): Rabbits were treated with 0.35 ml vehicle (40% propylene glycol, 10% ethanol in water).
- Group II (MLA): Rabbits were treated with MLA (35 μg/kg, i.v.) 24 h before I/R.
- Group III (MLA-5-HD): Rabbits were treated with MLA as in group II, and 24 h later, they were given 5-HD (5 mg/kg) 15 min before ischemia.
- Group IV (5-HD controls): Rabbits were treated with 5-HD 15 min before I/R.
All animals were subjected to ischemia by occlusion of the coronary artery for 30 min and reperfusion for 3 h.
Rabbits were anesthesized with i.m. ketamine HCl (35 mg/kg) and xylazine (5 mg/kg), and additional anesthesia was administered during the experiment as needed. A tracheotomy was performed, and the rabbits were ventilated by positive pressure with room air, supplemented with 100% oxygen if needed to maintain blood gases in physiologic range. The rate and volume of respiration were also adjusted during the experiment to maintain pH, pCO2, and pO2 in normal range. Catheters were placed in the left carotid artery and jugular vein for blood pressure measurements and arterial blood gas measurements, and drug and fluid administration, respectively. A left thoracotomy was performed between the fourth and fifth ribs, and the pericardium was opened to expose the heart. A 5-0 silk suture on a curved tapered needle was passed around the left coronary artery at approximately midway between the apex and base of the heart. The ends of the tie were passed through a small vinyl tube to form a snare. The coronary artery was occluded by pulling the snare tight and securing with a hemostat. Myocardial ischemia was determined by ST-segment elevation and the appearance of regional cyanosis (blanching). Reperfusion was documented by observation of hyperemia and resumption of contractions in the area below the snare on release.
Measurement of infarction and risk areas
At the end of each of the experiments, the ligature around the large marginal branch of the circumflex artery was retightened and ∼4 ml of 10% Evans blue dye was injected into the jugular vein until the eyes turned blue. The rabbits were killed, and their hearts harvested and cut into six transverse slices of equal thickness. The area at risk was determined by negative staining. The slices were stained by incubation for 15 min in 1% triphenyl tetrazolium chloride (TTC) in isotonic pH 7.4 phosphate buffer. Tetrazolium reacts with reduced nicotinamide adenine dinucleotide (NADH) in the presence of dehydrogenase enzymes, causing the viable tissue (area at risk) to stain a deep red color. The necrotic tissue does not react with TTC and remains a pale yellow color. After staining, the sections were placed in formalin for preservation, and measurements of risk area, infarct area, and left ventricle were made by using Bioquant imaging software for computer-aided morphometry. From each section, the ischemic risk area (unstained by blue dye) and the infarcted area (unstained by TTC) were outlined and measured by planimetry. The area from each region was averaged from the slices. Infarct size was expressed as percentage of the ischemic risk area.
Blood pH, gases, and hemodynamic measurements
Arterial pH, pO2, and pCO2 were measured at selected intervals by blood gas analyzer (Ciba Corning model 2380, Halstead, Essex, England) and maintained within a normal physiologic range (pH, 7.35-7.45; pO2, 90-110 mm Hg; pCO2, 35-45 mm Hg) by adjusting the respiratory rate and oxygen flow. Heart rate, mean arterial blood pressure, systolic blood pressure, and diastolic blood pressure were measured, and the rate-pressure product was calculated as the product of the heart rate and the systolic blood pressure.
Animals were omitted from the study if (a) coronary artery occlusion did not produce severe ischemia (i.e., >20% of the ventricle at risk); (b) ventricular fibrillation or severe hypotension was observed during the period of ischemia or reperfusion; or (c) the animal died during the surgical procedure and did not finish the entire protocol.
All measurements of infarct size and area at risk are expressed as group mean ± SEM. Changes in hemodynamics and infarct-size variables were analyzed by a two-way repeated measures analysis of variance (ANOVA). If the global tests showed major interactions, post hoc contrasts between different time points within the same group or different groups were performed by using Tukey's test. Statistical differences were considered significant if the p value was <0.05.
Hemodynamic data from the four experimental groups are shown in Table 1. Heart rate was comparable among the four groups at baseline and during I/R. Mean arterial blood pressure was not significantly different among the four experimental groups at all time points measured.
The area at risk, expressed as a percentage of the left ventricle (LV), and the infarct size, expressed as a percentage of the area at risk, are shown in Fig. 1. The percentages of myocardium at risk were comparable in all groups. The rabbits that received MLA-vehicle, 24 h before the 30-min period of coronary artery occlusion and 180 min of reperfusion, had a 40.5 ± 8.5% necrosis in the area at risk. This was not significantly different from the infarct size measured in rabbits that received 5-HD, 15 min before the period of sustained I/R. MLA administered 24 h before I/R resulted in significant reduction in the infarct size to 15.1 ± 1.4% (p < 0.05), as compared with the animals that were treated with either MLA-vehicle or 5-HD. The infusion of 5-HD, 15 min before the period of I/R, in the rabbits that were pretreated with MLA, completely abolished the myocardial protective effect of MLA with a infarct size of 51.8 ± 5.8% (p < 0.05 versus MLA group).
The purpose of this study was to determine whether the infarct-size reduction achieved by the administration of MLA 24 h before I/R could be blocked by 5-HD, an ischemia-selective, K-ATP channel antagonist that has no systemic metabolic effects. Our results showed that 5-HD has no proischemic effects, as judged by the infarct size after I/R, and it abolished the myocardial protective effect of MLA, thereby implicating the KATP channel in the mechanism of PC by MLA.
Recently it was demonstrated that ischemic PC produces delayed protection called the second window of protection (SWOP), which appears 24 h later (19,20). This form of protection is important because it is clinically relevant as compared with short-term PC, the effect of which lasts only for 90 min (21). However, the cardioprotective effect of SWOP is not so robust as short-term PC (i.e., 50 vs. 75-80%). Therefore novel pharmacologic agents that can produce longer-lasting superior protection after I/R are being investigated.
In our investigation, we used MLA, which has been demonstrated to provide powerful protective effects in many in vivo studies (9-12). MLA has been shown to exert dose-dependent protective effects on reduction of infarct size in the rabbit heart (22). The protective effects became apparent at 5 μg/kg, but significant reduction in infarct size was observed at a dose of 10 and 35 μg/kg. Yao et al. (12) showed that a 24-h pretreatment with MLA at a dose of 30-100 μg/kg resulted in a marked dose-dependent reduction in myocardial infarct size and that the beneficial effects were independent of changes in peripheral hemodynamics, area at risk, and collateral flow to the ischemic region. In our investigation, a 24-h period was allowed to elapse between MLA dosing and induction of regional ischemia, as this represents an intermediate time in the window of cardioprotection elicited by this agent. More specifically, in the rabbit-infarct model, a single intravenous dose of MLA (35 μg/kg) reduces infarct size significantly between 9 and 36 h, with protection dissipating by 48 h after treatment (data not shown).
The mechanism of myocardial protection by this promising drug continues to be an fascinating area of investigation, and accordingly, several novel possibilities have been evaluated. We first hypothesized that MLA could stimulate the synthesis of cytoprotective heat-shock proteins that might be responsible for delayed myocardial protection in vivo. Our data (23) as well as those of Baxter et al. (24), clearly demonstrated no increase in 70-kDa heat-shock protein despite significant reduction of infarct size in vivo. Mei et al. (13) first suggested that opening of ATP-sensitive potassium channel (KATP channel) was involved in cardiac protection in dogs because glibenclamide, a sulfonylurea KATP channel antagonist, abolished the cardioprotective effect of MLA. In addition, these investigators demonstrated dose-dependent shortening of action potential with MLA, which was completely abolished by glibenclamide. Recently Elliott et al. (22) further confirmed these observations by blocking MLA-induced protection in a rabbit model of I/R. The mechanism by which MLA opens KATP channel is not known. Bacterial endotoxins were demonstrated to increase free radical production in many different models (25,26), although their exact role is not known. MLA may induce PC by generation of free radicals, which have been shown to initiate late PC in myocytes (27). Endotoxin also was suggested to stimulate the synthesis of inducible NO synthase (iNOS; 28). Treatment with conventional endotoxin is known to increase the de novo synthesis of iNOS (29,30). The ability of endotoxin to induce iNOS has been shown in neonatal and adult cardiac myocytes (31-33). The administration of Escherichia coli endotoxin in rat was markedly antiarrhythmic (34), and this protection was abolished by pretreatment with dexamethasone, suggesting that the induction of iNOS may be a key event in the mediation of this protection. Because MLA is a nontoxic analog of endotoxin, it can possibly induce the generation of NO via increased activity or synthesis of the iNOS enzyme. Miyoshi et al. (35) demonstrated that the endotoxin-induced increased KATP channel activity of cells was blocked by iNOS inhibitor, suggesting a role of endogenous NO in the activation of the channel. Recent studies by Hu et al. (36) suggested that protein kinase C (PKC) activated the KATP channel in rabbit and human ventricular myocytes by reducing channel sensitivity to intracellular ATP. These investigators reported that phorbol 12,13-didecanoate induced activation of the KATP channel, which was blocked by highly selective PKC inhibitor. However, no studies are available to show direct activation of PKC with MLA.
Opening of the KATP channel has been shown to be protective because of the increase in the outward potassium current, resulting in the shortening of the action potential (37), which in turn may spare ATP, thereby allowing less entry of calcium into the myocyte through the voltage-sensitive calcium channel. Decreased intracellular calcium overload then results in a reduction of ischemic injury and therefore leads to better preservation of myocytes. Opening of the KATP channel has been implicated in the protection of myocardium after ischemic PC as well as drug-induced protection. There is some controversy surrounding the effectiveness of glibenclamide in blocking PC. Downey et al. (38) failed to block PC with glibenclamide in the rabbit. In addition, they observed a proischemic effect of glibenclamide (i.e., the infarcted area was increased in nonpreconditioned hearts). These authors argued that the anesthetic agent used may affect the ability of glibenclamide to block PC. They suggested that glibenclamide failed to block PC when pentobarbital (PTB) was used for anesthesia. Switching the anesthetic agent from PTB to ketamine-xylazine resulted in blockade of PC (39). In our study, we used ketamine-xylazine as the anesthetic agent, although we did not observe the proischemic effect of glibenclamide, because the infarct size was not significantly different between the glibenclamide-treated I/R hearts and the nontreated controls. Similarly, Gross and Auchampach (40) and Qian et al. (41) used PTB in their dog and rat studies and still were able to block the PC. In our studies, we used 5-HD, an ischemia-selective KATP channel antagonist, which has no systemic metabolic effects. Moreover, 5-HD is highly soluble in saline, whereas glibenclamide requires a mixture of polyethylene glycol, ethanol, and sodium hydroxide or DMSO to solubilize. Therefore vehicle effects of 5-HD are virtually nonexistent. Moreover, our results have shown that 5-HD has no proischemic effects, as judged by the infarct size after I/R and that it abolishes the myocardial protective effect of MLA, thereby implicating the K-ATP channel in the mechanism of protection.
Both 5-HD and glibenclamide were shown to he unable to reverse either the preischemic cardiodepression or the postperfusion cardioprotective effects of the calcium channel blocker diltiazem, thus suggesting that these drugs lack nonspecific proischemic actions (14,42,43). This was confirmed in the isolated rat-heart model of global ischemia by McCullough et al. (14), who found that that the administration of 5-HD or glibenclamide had no effect on the severity of the ischemia. In our studies, we used 5-HD, which has minimal or no pharmacologic activity in vascular smooth muscle or normal cardiac muscle (14). Whereas glibenclamide blocks the effect of the KATP channel activator cromakalim in rat aorta and in normal cardiac preparations, 5-HD was unable to modify the actions of cromakalim in either vascular smooth muscle or normal cardiac muscle (44-47). In addition, McCullough et al. (14) reported that whereas glibenclamide completely reversed the preischemic coronary vasorelaxant effect of cromakalim, 5-HD had no effect. The divergent actions of glibenclamide and 5-HD on the vascular and cardiac actions of cromakalim suggest that these two agents are active at different receptors or at different sites on the same receptor in normal and ischemic tissue.
Myocardial PC being a physiologically relevant mechanism that significantly limits the size of the infarct resulting from a subsequent sustained period of ischemia, its pharmacologic induction constitutes a promising new way of treating myocardial ischemia. Because MLA is safe and well tolerated in humans at physiologically active doses, our results suggest that it may have a role in the preservation of viable myocardium in situations in which induction of ischemia is inevitable, as in coronary angioplasty and coronary artery bypass surgery. Our investigation further confirms that the KATP channel is involved in the mechanism of pharmacologic PC with MLA in rabbits.
Acknowledgment: This work was supported in part by grants Hl 46763, 54045, and 07357 from the National Institutes of Health.
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