SPECIAL TOPIC
Glycolipid RC-552 induces delayed preconditioning-like effect via iNOS-dependent pathway in mice

Division of Cardiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298

	ABSTRACT

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We recently demonstrated that monophosphoryl lipid A (MLA)-induced delayed cardioprotection is mediated by inducible nitric oxide synthase (iNOS) in mice. In the present study, we determined whether RC-552, a novel synthetic glycolipid related in chemical structure to MLA, could afford similar protection. Adult mice were pretreated with vehicle or RC-552 (350 µg/kg ip, n = 7 mice/group) 24 h before global ischemia and reperfusion in a Langendorff isolated, perfused heart model. A group of RC-552-treated mice received S-methylisothiourea (SMT), a selective inhibitor of iNOS (3 mg/kg ip), 30 min before heart perfusion. Myocardial infarct size was significantly reduced from 19.2 ± 2.0% in vehicle to 8.2 ± 2.9% in RC-552 group (P < 0.05). Treatment with SMT abolished RC-552-induced reduction in infarct size (20.0 ± 3.9%). In addition, RC-552 failed to reduce infarct size in isolated hearts from iNOS knockout mice (27.1 ± 2.8%) compared with that in hearts from control knockout mice without drug treatment (22.9 ± 5.4%). Acute buffer perfusion with RC-552 (0.1, 1.0, or 2.5 µg/ml) for 8 min immediately before ischemia-reperfusion did not reduce infarct size significantly. We concluded that RC-552 induces delayed cardioprotection via an iNOS-dependent pathway.

ischemia-reperfusion; myocardial infarction; signal transduction; pharmacological preconditioning; monophosphoryl lipid A

	INTRODUCTION

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SEARCHING FOR new pharmacological agents with the ability to enhance myocardial endogenous protective mechanisms against ischemia-reperfusion injury has been the focus of cardiovascular research for over two decades (17, 18). Among a few agents that can potentially induce "pharmacological preconditioning" in myocardium against ischemia, monophosphoryl lipid A (MLA) has been extensively investigated (4). MLA is derived and purified from bacterial lipopolysaccharide (20). It retains the immunomodulatory properties of the parent endotoxin molecule without the associated toxicity. MLA maintains many of the beneficial immunologic activities of the parent molecule, leading to an enhanced tolerance to endotoxemia in both laboratory animals (6) and human subjects (1), possibly through cytokine production, macrophage stimulation, or other humoral and cell-mediated immune responses (1, 6, 10). Moreover, numerous studies have shown that MLA can also induce a remarkable anti-ischemic cardioprotection in various animal species, including dogs (15, 19, 30), pigs (32), rabbits (2, 5, 8, 31, 33), rats (14, 16, 21, 23), and mice (28). There is mounting evidence supporting the involvement of ATP-sensitive potassium (K_ATP) channels in the mechanism of protection with MLA (5, 8, 15). Most recently, we (28) and other investigators (23, 32, 33) have demonstrated that the MLA-induced delayed anti-ischemic cardioprotection is mediated by inducible nitric oxide synthase (iNOS).

RC-552 is a novel synthetic structural analog of MLA recently synthesized by Ribi ImmunoChem Research (Hamilton, MT). This drug retains the cardioprotective activity without possessing the ability to elicit cytokines or a pyrogenic effect even at a high-dose level. RC-552 is structurally distinct from the congeners found in the natural product MLA, incorporating a C-18 fatty ester at the -hydroxy functional group of the 2' amide (Fig. 1). In the present study we determined whether RC-552 could afford immediate and/or delayed anti-ischemic protection in the mouse heart. A possible role of iNOS in the underlying mechanisms of the RC-552-induced late cardioprotection was also investigated with the use of a selective iNOS inhibitor as well as iNOS gene knockout mice. The preliminary results of this study have been published in abstract form (29).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Chemical structure of RC-552.

	METHODS

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Animals

Adult male outbred ICR mice were supplied by Harlan Sprague Dawley (Indianapolis, IN), and the adult male F₂ homozygous (/) iNOS gene knockout B6,129 mice were purchased from Jackson Laboratory (Bar Harbor, ME). The mean body weight for these animals was 31.3 ± 0.5 g. When they arrived, the animals were allowed to readjust to the housing environment for at least 3 days before any experiment. Standard rodent food and water were freely accessible for the mice. All animal experiments were conducted under the guidelines for the humane use and care of laboratory animals for biomedical research as published by the National Research Council (revised 1996).

Drugs and Chemicals

RC-552 (lot no. 533-062) and its vehicle solvent were provided by Ribi ImmunoChem Research. RC-552 was produced through total organic synthesis according to the method of Johnson et al. (9). Specifically, R-3-dodecanoyloxytetradecanoyl was replaced as R₃ group of the intermediate compound 17 with R-3-octadecanoyloxytetradecanoyl. The free acid product was subsequently salted as the monobasic triethylamine salt to produce RC-552. The vehicle was composed of 40% propylene glycol and 10% ethyl alcohol in water. All other chemicals, including S-methylthiourea sulfate (SMT), a selective inhibitor of iNOS (22), were purchased from Sigma Chemical (St. Louis, MO).

Langendorff Isolated Perfused Heart Preparation

The methodology for the isolated, perfused mouse heart preparation was described previously in detail (26, 27). In brief, the animal was anesthetized with pentobarbital sodium (120 mg/kg) and heparin (33 units ip), and the heart was quickly removed from the thorax and placed into a small dish containing ice-cold perfusate with heparin. The aortic opening of the heart was rapidly cannulated and tied on a 20-gauge blunt needle that was, in turn, connected to a Langendorff perfusion system. After the cannulation, heart was retrogradely perfused at a constant pressure of 55 mmHg with modified Krebs-Henseleit solution containing (in mM) 118 NaCl, 24 NaHCO₃, 2.5 CaCl₂, 4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 11 glucose, and 0.5 EDTA. The perfusion solution was continuously gassed with 95% O₂-5% CO₂ (pH 7.34-7.49) and warmed by a heating/cooling bath. The heart temperature was continuously monitored and maintained at 37°C throughout the experiment. Ventricular function was measured by a force-displacement transducer (model FT03, Grass) attached to the apex with a no. 5 surgical thread and a rigid metal hook. The resting tension of the isolated heart was adjusted to ~0.30 g and kept without readjustment thereafter. Ventricular developed force was recorded with the use of a Beckman R-511A polygraph that was connected to the force transducer and calibrated before each experiment. Coronary flow rate was calculated by timed collection of the perfusate. The hearts were not paced.

Drug Pretreatment and Ischemia-Reperfusion Protocols

"Delayed window" study. Mice were treated with either RC-552 (350 µg/kg ip) or volume-matched vehicle. The hearts were isolated 24 h later and, after a 30-min stabilization period, subjected to 20 min of no-flow normothermic global ischemia and 30 min of reperfusion. A group of RC-552-treated mice also received SMT (3 mg/kg ip), the iNOS inhibitor, 30 min before heart perfusion. Two additional groups of iNOS knockout mice were subjected to the same ischemia-reperfusion protocol with or without pretreatment with RC-552. The experimental groups and protocol of this study are shown in Fig. 2.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Experimental protocol for "delayed window" study consisting of 5 experimental groups (n = 7 mice/group). Vehicle group was pretreated with vehicle (intraperitoneally) 24 h before ischemia-reperfusion (I/R). RC-552 group was pretreated with RC-552 (350 mg/kg ip) 24 h before I/R. RC-552 + SMT group was pretreated with RC-552 24 h before I/R, with S-methylisothiourea (SMT, 3 mg/kg ip) given 30 min before IS/R. iNOS-KO group was inducible nitric oxide synthase (iNOS) knockout mice subjected to I/R without pretreatment. RC-552 + iNOS-KO group was iNOS knockout mice pretreated with RC-552 24 h before I/R.

"Early window" study. Nontreated mouse hearts were isolated and stabilized for 20 min before the baseline functional data were collected. The hearts then underwent intracoronary perfusion with RC-552 (0.1, 1.0, or 2.5 µg/ml) or vehicle for 8 min through a side arm of the three-way stopcock connected directly above the aortic cannula with a Harvard microdialysis syringe pump (model 22). The pump speed was set at 0.25 ml/min, which was equivalent to ~15% of the normal coronary flow rate of the heart. After the postdrug functional parameters within 1 min were measured, the hearts were subjected to the ischemia-reperfusion protocol as described in the delayed window study. The experimental groups and protocol are shown in Fig. 3.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Experimental protocol for "early window" study consisting of 4 groups (n = 4-6 mice/group). Vehicle perfusion group received intracoronary perfusion with vehicle at flow rate of 0.25 ml/min for 8 min immediately before I/R. Three RC-552-perfused groups received intracoronary perfusion with RC-552 in 3 different drug doses (i.e., 0.1, 1.0, or 2.5 µg/ml, respectively) at a flow rate of 0.25 ml/min for 8 min immediately before I/R.

Exclusion Criteria

The hearts were excluded from further data analysis in the following undesirable situations: 1) time delay in the aortic cannulation (>3 min); 2) damage to the aorta during the cannulation; 3) sustained arrhythmia during the first 20 min of stabilization; or 4) depressed ventricular developed force (<0.1 g) at the end of stabilization.

Measurement of Infarct Size

At the end of each experiment, the heart was immediately removed from the Langendorff apparatus, weighed, and frozen at 20°C. The frozen heart was manually cut into seven to eight transverse slices of approximately equal thickness (~0.8 mm) and stained by incubation in 10% triphenyltetrazolium chloride (TTC) for 30 min at room temperature (~22°C). TTC buffer was then replaced with 10% formaldehyde, and the slices were fixed for 4-6 h before the infarct area and risk zone were measured using computer morphometry (Bioquant 98). The risk area was calculated as total ventricular area minus the area of cavities. The infarct size was calculated as a percentage of the risk area.

Data Analysis and Statistics

Each experimental group consisted of four to seven animals. The group means and their standard errors (SE) for each parameter are presented. One-way ANOVA was used to compare the values of three or more groups. If a significant value of F was obtained, the Student-Newman-Keuls post hoc test was subsequently used to make pairwise comparisons among the groups. A paired t-test was also used to compare any pre- and posttreatment values for any given parameter. P < 0.05 was considered statistically significant.

	RESULTS

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Exclusions

A total of 61 hearts were subjected to ischemia-reperfusion protocols in the delayed window (n = 7 mice/group, Fig. 2) and early window studies (n = 4-6 per group, Fig. 3) for the assessment of ventricular function as well as infarct size. Among them, seven hearts (i.e., 11% of the 61 perfused hearts) were excluded according to the exclusion criteria described in METHODS (3 hearts because of delay in cannulation time, 3 because of aortic damage, and 1 because of depressed ventricular force).

Cardiac Hemodynamic and Contractile Function: Delayed Window Study

There was no significant difference in the preischemic basal value of ventricular contractile parameters (i.e., developed force, rate-force product, and resting tension) among the five experimental groups (Table 1). Pretreatment with RC-552 resulted in a moderate increase in preischemic basal coronary flow rate (2.99 ± 0.35 ml/min) compared with 2.21 ± 0.30 in the vehicle group, although this change was not statistically significant (P > 0.05). After 20 min of global ischemia, ventricular developed force and rate-force product were significantly depressed in all experimental groups, regardless of the pretreatment conditions during reperfusion (Table 1). The resting tension and heart rate were not significantly different between the preischemic and reperfusion periods (Table 1). Postischemic coronary flow was not significantly different from its preischemic values for all of the groups except the RC-552 group, in which coronary flow was significantly reduced to 2.01 ± 0.21 ml/min from the corresponding preischemic value (P < 0.05). The coronary flow was not higher in RC-552-treated groups compared with the vehicle group.

View this table: [in this window] [in a new window]   Table 1.   Cardiac functional parameters in the "delayed window" study

Infarct Size: Delayed Window Study

Myocardial infarction was evident in the vehicle-treated mouse hearts after ischemia-reperfusion (19.2 ± 2.0% of risk zone, Fig. 4A). Pretreatment with RC-552 caused a significant reduction in infarct size (8.2 ± 2.9%, P < 0.05) that was blocked by pretreatment with SMT (20.0 ± 3.9%). RC-552 treatment in the iNOS knockout mice failed to reduce infarct size after ischemia-reperfusion (27.1 ± 2.8%) compared with the control knockout mice without drug treatment (22.9 ± 5.4%). The area at risk was not different among the groups (Fig. 4B). Representative samples of the TTC-stained heart slices are shown in Fig. 5. The viable (red-stained area) and necrotic tissues (pale area) are clearly distinguishable.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of RC-552 on infarct size (A), expressed as a percentage of risk area (B), in delayed window study. * P < 0.05 vs. all other groups.

View larger version (117K): [in this window] [in a new window]   Fig. 5.   Representative photographs of triphenyl tetrazolium chloride-stained mouse heart slices demonstrating infracted (pale) and viable (red) areas in delayed window study. A: vehicle group; B: RC-552 group; C: RC-552 + SMT group; D: RC-552 + iNOS-KO group.

Cardiac Hemodynamic and Contractile Function: Early Window Study

There was no significant difference in the basal functional parameters (i.e., developed force, rate-force product, and resting tension) among the groups after 20 min of stabilization (Table 2). Intracoronary perfusion with vehicle or low-dose RC-552 (0.1 µg/ml) for 8 min did not cause a significant change in ventricular function. However, perfusion with higher doses of RC-552 (1.0 or 2.5 mg/ml) immediately resulted in a consistent depression of ventricular contractile function (P < 0.05) without significant effect on the heart rate (Table 2). Paradoxically, after 20 min of global ischemia, ventricular developed force and rate-force product were well preserved in the vehicle-perfused group but remained depressed in all three RC-552-perfused groups during reperfusion (Table 2). The resting tension was not significantly different among the groups, whereas heart rate appeared to be higher in the low-dose RC-552-perfused group but lower in the high-dose RC-552-perfused group (P > 0.05, Table 2). Postischemic coronary flow was slightly higher in the low-dose RC-552-perfused group compared with all other groups, although the difference was not statistically significant (P > 0.05).

View this table: [in this window] [in a new window]   Table 2.   Cardiac functional parameters in the "early window" study

Infarct Size: Early Window Study

Myocardial infarction was observed in all of the four experimental groups after ischemia-reperfusion (Fig. 6A). In contrast to the results in the delayed window study, intracoronary perfusion with the three different doses of RC-552 before ischemia-reperfusion caused only a marginal reduction in infarct size (P > 0.05). The area at risk for the globally ischemic hearts was not different among the groups (Fig. 6B).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Acute effect of intracoronary infusion of RC-552 on infarct size (A), expressed as a percentage of risk area (B), in mouse heart. No significant difference among groups was observed (P > 0.05).

	DISCUSSION

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Salient Findings

The goal of the present investigation was to demonstrate the early and delayed cardioprotective effects of the novel glycolipid RC-552 and to delineate the underlying mechanism of protection. Our results show that pretreatment with RC-552 (350 µg/kg ip) 24 h before ischemia-reperfusion resulted in a significant reduction in myocardial infarct size. The anti-infarct effect was not associated with the improvement in postischemic ventricular contractile function or coronary flow. The RC-552-induced delayed cardioprotection was abolished by SMT and was completely absent in iNOS knockout mice. Intracoronary perfusion with RC-552 immediately before the global ischemia-reperfusion did not cause a reduction in infarct size. To our knowledge, this is the first study demonstrating the delayed cardioprotective effect of the novel synthetic glycolipid RC-552, which depends on iNOS in the isolated perfused mouse heart.

Our findings are in accord with the preliminary results from other laboratories demonstrating a similar delayed cardioprotection induced by intravenous pretreatment of RC-552 in vivo canine models (12, 24, 25). However, in contrast to the recent preliminary reports by Weber et al. (24, 25), we did not find early phase protection by intracoronary perfusion of RC-552 (Fig. 6). In fact, perfusion with higher doses of RC-552 (1.0 or 2.5 µg/ml) severely depressed ventricular function (Table 2). The postischemic cardiac function in the RC-552-perfused groups was significantly depressed compared with that in the vehicle-perfused group (Table 2). The exact reasons for this discrepancy are still unclear. Different animal species (mouse vs. dog) and experiment models (isolated heart vs. in situ heart) may account for the differences in the early window results obtained with RC-552.

Role of iNOS

Nitric oxide is critical in the signal transduction of ischemic myocardium (13). It has also been appreciated as the key trigger and mediator for the delayed phase of ischemic preconditioning (3). Its biological action can be cardioprotective against ischemia-reperfusion injury through coronary vasodilatation or reduction in myocardial oxygen consumption as well as opening of the K_ATP channels (28). Previous studies demonstrated that the cardioprotective effect of the first-generation glycolipid MLA was abolished by the iNOS inhibitor aminoguanidine in the rabbit infarct model (33). In addition, iNOS mRNA was induced between 4 and 8 h after administration of cardioprotective doses of MLA in rat and pig myocardium (23, 32). Moreover, we recently showed that MLA reduced infarct size in mice after global ischemia-reperfusion and that anti-infarct protection was absent in the iNOS gene knockout mice (28). Therefore, we hypothesized that the second-generation glycolipid RC-552 might also induce delayed cardioprotection in the mouse heart via an iNOS-sensitive mechanism. In the present investigation, RC-552-induced protection was absent in the iNOS knockout mice, suggesting an obligatory role of this isoform in the protective process. It is interesting to compare our present findings with those recently reported by Imagawa et al. (7), who suggested iNOS as a mediator of delayed ischemic preconditioning because the iNOS inhibitors dexamethasone or aminoguanidine were able to abrogate the delayed infarct reduction afforded by ischemic preconditioning in the in situ rabbit hearts. Considering the ability of MLA and RC-552 to activate the iNOS-dependent signal transduction pathway, one could assume that these drugs may be useful in inducing the delayed preconditioning-like effect and mimicking pharmacologically the powerful protection accorded with ischemic preconditioning.

In the present study, we observed some similarities as well as differences in the cardioprotective effects of RC-552 versus MLA. First, both drugs caused a remarkable reduction in infarct size after ischemia-reperfusion 24 h after the administration of an identical drug dose (350 µg/kg ip, Fig. 4) without any significant changes in the postischemic contractile function (Table 1) (28). Second, iNOS is the common mediator for the delayed cardioprotection induced by either MLA or RC-552. On the other hand, the delayed vascular effects of RC-552 were less obvious than those of MLA. Unlike MLA, which induced significant improvement in both pre- and postischemic coronary flow (28), RC-552 caused only a mild increase in the preischemic coronary flow and had no effect on the postischemic coronary flow. These data suggest that a vascular-related mechanism may play a minor role in RC-552-induced protection. Another difference was that the iNOS inhibitor SMT completely abolished the RC-552-induced antinecrotic protection, whereas MLA-induced protection was partially blocked (28). Therefore, it appears that MLA may have activated other mediators of delayed protection in addition to iNOS. We speculated that MLA may have stimulated endothelial constitutive NOS in the heart and improved vascular endothelial function independent of the iNOS enzyme (28). The present study suggests that the iNOS-independent effects were less distinct in the RC-552-induced late cardioprotection. Because the role of constitutive forms of NOS in RC-552-induced protection is still not clear, further investigations are necessary to answer this question.

In conclusion, our results demonstrated that the novel synthetic glycolipid RC-552 induced a delayed preconditioning-like cardioprotection against myocardial infarction in the ischemic mouse heart. The RC-552-induced cardioprotection was mediated by iNOS because the protective effect was blocked by selective inhibition of this isoform of the enzyme and was completely absent in the iNOS knockout mice. The delayed cardioprotective effects afforded by RC-552 as well as MLA may represent a unique pharmacological preconditioning approach that could lead to development of this drug to protect the heart from lethal ischemic attack.

	ACKNOWLEDGEMENTS

We thank Drs. G. T. Elliott and P. Weber, Ribi ImmunoChem Research, Hamilton, MT, for kindly providing the new drug RC-552 and for advice on performing this study.

	FOOTNOTES

This work was funded in part by National Heart, Lung, and Blood Institute Grants HL-51045 and HL-59469 (to R. C. Kukreja). L. Xi was supported by a Research Fellowship from the American Heart Association, Mid-Atlantic Affiliate (F98273V).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. C. Kukreja, Division of Cardiology, Box 980281, Medical College of Virginia, Richmond, VA 23298 (E-mail: rakesh{at}hsc.vcu.edu).

Received 30 July 1999; accepted in final form 27 August 1999.

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