Tyrosine kinase signaling in action potential shortening and expression of HSP72 in late preconditioning.

We investigated the role of tyrosine kinase (TK) signaling in the opening of the ATP-sensitive K(+) (K(ATP)) channel and 72-kDa heat shock protein (HSP72) expression during late preconditioning. Rabbits were subjected to surgical operation (sham) or were preconditioned (PC) with four cycles of 5 min of ischemia and 10 min of reperfusion. Twenty-four hours later, animals were subjected to 30 min of ischemia and 180 min of reperfusion. Genistein (1 mg/kg ip) was used to block the receptor TK. Six groups were studied: control, sham, genistein-sham, PC, genistein-PC, and vehicle-PC group (1% dimethyl sulfoxide). Genistein or vehicle was given 30 min before the surgical procedure. Genistein pretreatment decreased the expression of HSP72 in PC hearts and suppressed action potential duration shortening during ischemia in sham and PC groups. Infarct size (%risk area) was reduced in the PC (11.6 +/- 1.0%) and vehicle-PC (19.3 +/- 2.0%) compared with the control (40.0 +/- 3.8%) or sham (46.0 +/- 2.0%) groups (P < 0.05). Genistein pretreatment increased infarct size to 46.4 +/- 4.1% in the PC hearts. We conclude that TK signaling is involved in K(ATP) channel opening and HSP72 expression during late PC.

Preconditioning of swine heart with monophosphoryl lipid A improves myocardial preservation.

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 microg/kg); group IV, MLA (35 microg/kg); and group V, MLA and AMG (35 microg/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.

The novel glycolipid RC-552 attenuates myocardial stunning and reduces infarct size in dogs.

The novel glycolipid RC-552 shares common structural features with the natural products lipid A and the previously described cardioprotectant monophosphoryl lipid A. RC-552 administered to dogs as a bolus intravenous dose (35-70 microg/kg) either 24 h or 10 min prior to 60 min of regional myocardial ischemia and 3 h of reperfusion significantly (P<0.05 v control) reduced infarct size (IS) as assessed by triphenyltetrazolium staining from 27.0+/-2.3% of the area-at-risk (AAR) to 13.3+/-2.2% and 15.0+/-3.0%, respectively. Administration of the non-specific inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (30 mg/kg, subcutaneously) 1 h prior to ischemia blocked the ability of RC-552 (35 microg/kg, 24 h pretreatment) to reduce infarct size. Intravenous pretreatment with RC-552 (35 microg/kg) either 24 h or 10 min prior to five 5 min repetitive cycles of ischemia and reperfusion significantly improved regional myocardial segment shortening (percentage of control) at all time points during 2 h of reperfusion in dogs. These effects of RC-552 in either cardiac injury model occurred independent of differences in AAR, transmural blood flow during ischemia or hemodynamics throughout the experiment. In contrast with monophosphoryl lipid A (MLA), which has also been reported to be cardioprotective at similar doses in dogs, RC-552 was approximately 100 times less prone to cause fever in the USP rabbit pyrogen test. Likewise, RC-552 did not induce secretion of the proinflammatory cytokines TNF, IL-6 or IL-8 from THP-1 cells or alter the expression of adhesion molecules on human neutrophils at concentrations up to 10 microg/ml. MLA was active in these systems at concentrations in the range 0.1-1.0 microg/ml. In conclusion, RC-552 reduces myocardial infarct size and stunning in dogs in the absence of residual immunomodulatory activity.

Monophosphoryl lipid A: a novel nitric oxide-mediated therapy to attenuate platelet thrombosis?

Nitric oxide (NO) is a potent inhibitor of platelet aggregation. However, the benefits of NO-based therapies can be confounded by concomitant hypotension. Monophosphoryl lipid A (MLA) is a nontoxic derivative of endotoxin that purportedly increases nitric oxide synthase (NOS) activity and, presumably, NO production, yet has a hemodynamically benign profile. Thus our aims were to determine whether (a) MLA attenuates in vivo platelet aggregation in damaged and stenotic canine coronary arteries by a NO-mediated mechanism but without reductions in arterial pressure; and (b) the platelet inhibitory effects are manifest in vitro. To address the first aim, anesthetized dogs underwent coronary injury + stenosis, resulting in cyclic variations in coronary blood flow (CFVs) caused by the formation/dislodgement of platelet-rich thrombi. In protocol I, dogs received MLA (100 microg/kg + 40 microg/kg/h) or vehicle beginning 15 min before stenosis. Protocol II was identical, except the NOS inhibitor aminoguanidine was coadministered with MLA/vehicle. Coronary patency was assessed throughout the initial 3 h after injury + stenosis. Infusion of MLA did not result in hypotension. However, in protocol I, the median nadir of the CFVs was higher (2.1 vs. 0.8 ml/min; p < 0.05), median duration of total thrombotic occlusion tended to be reduced (0 vs. 10.4 min; p = 0.1), and mean flow-time area, expressed as a percentage of baseline flow, was increased (53 +/- 9% vs. 33 +/- 3%; p < 0.05) in MLA-treated versus vehicle-treated dogs. In contrast, in protocol II, vessel patency was comparable in both groups. Finally, whole blood impedance aggregometry (protocol HI) revealed a significant reduction in the in vitro platelet aggregation in blood samples receiving exogenous MLA, which was blocked by coadministration of exogenous aminoguanidine. Thus MLA attenuates platelet-mediated thrombosis in both damaged and stenotic canine coronary arteries and in vitro, possibly by an NO-mediated mechanism, but without concomitant hypotension.

Cellular mechanisms of infarct size reduction with ischemic preconditioning. Role of calcium?

Brief episodes of ischemia protect or "precondition" the heart and reduce infarct size caused by a subsequent sustained ischemic insult. Despite a decade of intensive investigation, the cellular mechanism(s) responsible for this paradoxical protection remain poorly understood. In this review, we focus on the emerging concept that alterations in intracellular calcium homeostasis may participate in either triggering and/or mediating infarct size reduction with preconditioning.

Pharmacologic enhancement of tolerance to ischemic cardiac stress using monophosphoryl lipid A. A comparison with antecedent ischemia.

In comparison with ischemic preconditioning, MLA-mediated cardioprotection seems to show numerous common features. Like ischemia, MLA induces a first and second window (biphasic profile) of heightened tolerance to ischemia. As with delayed ischemic preconditioning, MLA protects against infarction, stunning, and arrhythmias associated with ischemia-reperfusion. In contrast with acute ischemic preconditioning, MLA reduces infarction and stunning. A role has been demonstrated for nitric oxide synthase and KATP channel activation in the mechanism of delayed preconditioning induced by ischemia and by MLA. Regarding acute preconditioning, kinase and KATP channel activation have been implicated as involved in the mechanism of ischemic preconditioning and also in MLA cardioprotection. Use of MLA or related compounds as cardioprotectants may represent a method for inducing acute tolerance to ischemia-reperfusion injury manifested as infarction or stunning, with the added benefit of a sustained delayed cardioprotective state being achieved.

Additive effects of late preconditioning produced by monophosphoryl lipid A and the early preconditioning mediated by adenosine receptors and KATP channel.

BACKGROUND: The cardioprotective effect of preconditioning can be exerted within 1 to 2 hours after initial ischemia, termed classical or early preconditioning, or can reappear 24 hours later as second window or late preconditioning. The objective of this study was to study the interaction between late and early preconditioning and to determine the potential underlying mechanism. METHODS AND RESULTS: Adenosine receptor agonists and a KATP channel opener were used to achieve early preconditioning, and Monophosphoryl lipid A (MLA) was used to induce late preconditioning. Cultured chick ventricular myocytes were used as a myocyte model of simulated ischemia and preconditioning. Prior treatment of the myocyte with MLA caused a dose-dependent decrease in the ischemia-induced myocyte injury 24 hours later, consistent with a late preconditioning effect. L-NMMA, glibenclamide, or 5-hydroxydecanoic acid administered during the ischemia blocked the MLA effect. Twenty four hours after MLA treatment, a 5-minute exposure to ischemia, adenosine, adenosine A1 agonist CCPA, or A3 agonist resulted in less myocyte injury during the subsequent prolonged ischemia, as compared with cells pretreated with the vehicle and subsequently exposed to the same early preconditioning stimuli. In addition to its ability to enhance the early preconditioning effect by A1 and A3 agonists, MLA pretreatment also increased the phorbol ester- and pinacidil-mediated early preconditioning effect. CONCLUSIONS: This study defined a novel interaction in which the cardioprotective effect of early preconditioning is additive to that of late preconditioning and raised the possibility that both agents can be used as combined therapy in the treatment of ischemic heart disease.

Induction of iNOS gene expression by monophosphoryl lipid A: a pharmacological approach for myocardial adaptation to ischemia.

Using the concept that exposing a cell to an adverse environment (stress) results in the stimulation of its endogenous defense system, hearts have been adapted to ischemia by exposing them to diverse stresses. Recently, 24-h pretreatment of monophosphoryl lipid A (MLA), a chemically modified derivative of endotoxin, was found to render the hearts more tolerant to ischemic reperfusion injury. Since nitric oxide has recently been implicated in myocardial preservation and since inducible nitric oxide synthetase (iNOS) was originally characterized in macrophages and shown to be maximally induced by bacterial lipopolysaccharides (endotoxin), we sought to determine whether MLA mediates its cardioprotective effects through the iNOS expression. For this, rats were injected with MLA (300 micrograms/kg) or vehicle (control), and after 24 h the animals were sacrificed and the isolated working hearts were made ischemic for 30 min followed by 30 min of reperfusion. MLA-treated hearts were found to be tolerant to ischemic reperfusion injury as evidenced by improved postischemic ventricular recovery. After 30 min of reperfusion, left ventricular developed pressure (LVDP) and its maximum first derivative (LVmaxdp/dt) were 13.3 +/- 0.3 kPa and 537 +/- 13 kPa/s, respectively, in the MLA-treated group, as compared with 10.2 +/- 0.4 kPa (p < 0.05) and 447 +/- 11 kPa/s (p < 0.05), respectively, for the control group. Aortic flow and coronary flow were 20.1 +/- 1.4 ml/min and 19.1 +/- 0.8 ml/min, respectively, in the MLA group, as compared with 9.5 +/- 0.8 ml/min (p < 0.05) and 15.9 +/- 0.7 ml/min (p < 0.05), respectively, for the untreated group. To examine the induction of the iNOS expression, RNAs were extracted from the control and MLA-treated hearts (after 2, 4, 6, 8, 12 and 24 h of treatment) and Northern blot analysis was performed using specific cDNA probe for iNOS. A single band of approximately 4.6 kb corresponding to iNOS mRNA was detected after 4 h of MLA treatment, while the maximal iNOS expression was found between 6-8 h of MLA treatment. The results of this study demonstrate that MLA induces the expression of iNOS and protects the myocardium from ischemic reperfusion injury.

Pharmacologic preconditioning with monophosphoryl lipid A is abolished by 5-hydroxydecanoate, a specific inhibitor of the K(ATP) channel.

We sought to determine the role of opening of adenosine triphosphate (ATP)-sensitive potassium channel (K(ATP) channel) in monophosphoryl lipid A (MLA)-induced myocardial protection after ischemia/reperfusion (I/R) in rabbit. We used 5-hydroxydecanoate (5-HD), an ischemia-selective inhibitor of K(ATP) channel, to block MLA-stimulated cardiac protection. Four groups of rabbits were studied: group I, MLA-vehicle; group II, MLA; group III, MLA + 5-HD; and group IV, 5-HD only. MLA (35 microg/kg, i.v.) or vehicle were given 24 h before I/R. 5-HD (5 mg/kg) was given 15 min before ischemia. All rabbits underwent 30-min coronary occlusion, followed by 3-h reperfusion. Area at risk was delineated by injection of Evan's blue, and infarct size was determined by tetrazolium staining. Pretreatment with MLA reduced infarct size (percentage of area at risk) from 40+/-8.6% to 15.1+/-1.5%. The infarct size increased to 51.9+/-5.8% with 5-HD in MLA-treated rabbits. 5-HD did not alter infarct size significantly when given in vehicle-treated control rabbits. These data suggest that MLA exerts its protective effect through activation of K(ATP) channel.

Antiischemic effect of monophosphoryl lipid A in conscious rabbits with hypercholesterolemia and atherosclerosis.

We studied whether monophosphoryl lipid A (MLA), an endotoxin derivative, protected the heart from planned ischemia in hypercholesterolemic conscious rabbits. Normal and hypercholesterolemic (8-week exposure to 1.5% cholesterol-enriched diet) conscious rabbits with right ventricular electrode and left ventricular polyethylene catheters were subjected to ventricular overdrive pacing (VOP: 500 beats/min over 10 min = control VOP). The resulting intracavitary ST-segment elevation, increase in left ventricular end-diastolic pressure (LVEDP), and a reduction of ventricular effective refractory period (VERP) were measured. Three days later the animals were given a single intravenous bolus of 10 or 30 microg/kg MLA or its solvent or both, and a second VOP (test VOP) was applied 24 h later. MLA decreased ST elevation and LVEDP increase from 2.1 +/- 0.16 to 1.27 +/- 0.25 and 0.97 +/- 0.13 mV and 14.6 +/- 1.2 to 11.1 +/- 1.0 and 12.4 +/- 1.2 mm Hg in normal animals and from 2.55 +/- 0.14 to 1.31 +/- 0.12 and 0.96 +/- 0.30 mV and from 21.0 +/- 1.6 to 11.7 +/- 1.3 and 12.4 +/- 1.3 mm Hg in atherosclerotic animals after 10- and 30-microg/kg doses, respectively (p < 0.001 for each). VOP-induced VERP reduction was also significantly alleviated by both MLA doses; nevertheless, 30-microg/kg MLA significantly prolonged resting VERP with a slight VERP reduction in response to pacing in both normal and atherosclerotic animals. We conclude that MLA produces a delayed antiischemic effect in both normal and hypercholesterolemic/atherosclerotic conscious rabbits.

Monophosphoryl lipid A attenuates myocardial stunning in dogs: role of ATP-sensitive potassium channels.

Results of previous studies indicate that monophosphoryl lipid A (MLA) reduces myocardial infarct size when administered 24 but not 1 h before a prolonged period of regional ischemia in dogs and rabbits. This cardioprotective effect of MLA could be reversed by the administration of the adenosine triphosphate (ATP)-sensitive potassium channel (K(ATP)) blockers, glibenclamide, or 5-hydroxydecanoate. MLA also was shown to attenuate myocardial stunning in dogs; however, its mechanism in this model remains unknown. Therefore the major aim of our study was to determine the dose-related effect of MLA to enhance contractile function in stunned myocardium and to determine the role of the K(ATP) channel in mediating its cardioprotective effect. To produce myocardial stunning, barbital-anesthetized dogs were subjected to five cycles of 5 min of left anterior descending (LAD) coronary artery occlusion interspersed with 10 min of reperfusion and finally followed by 2 h of reperfusion. Regional segment shortening (%SS) was determined by sonomicrometers implanted in the subendocardium of the ischemic region. Single intravenous doses of MLA in the range of 10-35 microg/kg given 24 h before ischemia resulted in an improvement in %SS over a 2-h reperfusion period. Similar to results obtained in the canine and rabbit infarct models, cardioprotection against stunning with MLA appears to require activation of K(ATP) channels during ischemia, because glibenclamide (50 microg/kg, 15 min before ischemia) completely blocked the effect of MLA to improve regional %SS during reperfusion. Cardioprotective doses of MLA were without effect on systemic hemodynamics, blood gases, and pH throughout the experiment. No treatment-related effects on regional myocardial blood flow were observed during ischemia or reperfusion. These results suggest that MLA improves %SS at doses of 10-35 microg/kg by an ATP-sensitive potassium channel-dependent process, and that MLA may mimic the antistunning effects observed during the second window of ischemic preconditioning.

Preconditioning of rat heart with monophosphoryl lipid A: a role for nitric oxide.

Preconditioning with monophosphoryl lipid A (MLA) protects rabbit hearts from prolonged ischemic reperfusion injury by a mechanism involving inducible nitric oxide synthase (iNOS) activation. This study was undertaken to determine whether MLA also could precondition rat hearts in a similar manner. Rats were injected with two different doses of MLA (300 microg/kg or 450 microg/kg i.v.) or vehicle (control), and after 24 hr the animals were sacrificed for preparation of isolated perfused rat hearts. Hearts were then perfused by working mode, and then made ischemic for 30 min followed by 30 min of reperfusion. Another group of hearts were treated simultaneously with a nitric oxide (NO) blocker, L-nitro-arginine-methyl-ester (L-NAME) (10 mg/kg) and MLA (450 microg/kg). For arrhythmia studies, 12 hearts were used in each group (total, 48 hearts). Cardiac functions were examined in a separate group of 24 hearts (n = 6/group). MLA-treated hearts (either dose) were tolerant to ischemic reperfusion injury as evidenced by improved postischemic ventricular recovery [coronary flow (ml/min) 19.1 +/- 0.8 (300 microg/kg MLA), 22.6 +/- 1.0 (450 microg/kg MLA) vs. 15.9 +/- 0.7 (control); aortic flow (ml/min) 20.7 +/- 1.8 (300 microg/kg MLA), 25.8 +/- 1.4 (450 microg/kg MLA) vs. 11. 0 +/- 0.8 (control); left ventricular developed pressure (kPa) 13.3 +/- 0.6 (300 microg/kg MLA), 14.6 +/- 0.2 (450 microg/kg MLA) vs. 10. 3 +/- 0.7 (control)]. Incidences of ventricular fibrillation and ventricular tachycardia were decreased compared with the control group only in the 450 microg/kg dose of MLA-treated hearts (92% to 33%). Pretreatment of the hearts with L-NAME inhibited the preconditioning effect of MLA. To examine the induction of the iNOS expression, RNAs were extracted from the control and MLA-treated hearts (after 2, 4,6, 8, 12 and 24 hr of treatment) and Northern blot analyses were performed with a specific cDNA probe for iNOS. A single band of approximately 4.6 kb corresponding to iNOS mRNA was detected after 4 hr of MLA treatment, whereas the maximal iNOS expression was found between 6 and 8 hr of MLA treatment. The results of this study demonstrated that MLA induced the expression of iNOS and protected the myocardium from ischemic reperfusion injury which is blocked by an inhibitor of NO synthesis, which suggests a role of NO in MLA-mediated cardioprotection.

Monophosphoryl lipid A induces delayed preconditioning against cardiac ischemia-reperfusion injury.

Monophosphoryl lipid A represents a novel agent capable of enhancing myocardial tolerance to ischemia/reperfusion injury. This cardioprotective activity of MLA manifests itself as a reduction in infarct size, myocardial stunning and dysrhythmias in multiple animal species. The drug appears to be efficacious in dogs and rabbits at doses of 10-35 micrograms/kg, with larger doses seemingly required in the rat. In the rabbit infarct model, protection appears 6 h following drug administration and lasts for 36 h. Although multifactorial mechanisms of ischemic tolerance may be induced by MLA, current evidence suggests that MLA's cardioprotective effects involve myocardial iNOS enzyme activation with nitric oxide coupled activation of myocardial KATP channels upon ischemic challenge. Monophosphoryl lipid A is presently being evaluated in Phase 2 clinical trials in patients undergoing cardiopulmonary bypass associated with coronary artery bypass engraftment or aortic valve replacement or reconstruction. Severity of lethal and reversible myocardial injury and dysrhythmia are study endpoints. Although further clinical testing will establish the utility of MLA as a cardioprotectant against ischemia/reperfusion injury in the human, presently this agent is proving very useful in expanding our understanding of mechanisms responsible for delayed cardiac preconditioning against ischemia/reperfusion injury.

Monophosphoryl lipid A protects adult rat cardiac myocytes with induction of the 72-kD heat shock protein: a cellular model of pharmacologic preconditioning.

We examined the in vitro preconditioning effect of non-toxic derivative of endotoxin, monophosphoryl lipid A (MLA) in adult rat cardiac myocytes. Cultured 5-7-day-old myocytes were preconditioned for 4 h by treatment with 200 ng/ml MLA. Twenty h later, cells were subjected to simulated ischemia by incubation in 0.75 mm sodium hydrosulfite, 12 mM KCl, 20 mM dl-lactic acid and 10 mM 2-deoxy-D-glucose (pH 6.5) for 2 h. MLA caused a significant reduction in the levels of LDH from 286+/-8 units/l in controls to 165+/-5 units/l (mean+/-s.e.m.; P<0.0001). Similarly, CK significantly decreased from 104+/-3.1 in controls to 85+/-1.4 U/l (P<0.001). Western blot analysis indicated a significant accumulation of 72 kD heat shock protein in MLA treated as compared to control cells. No changes in 27, 32, and 90 kD heat shock proteins were discernible in the MLA treated group. These data suggest a significant "anti-ischemic" effect of MLA in myocytes that is accompanied by induction of 72 kD heat shock protein.

Disparate effects of preconditioning and MLA on 5'-NT and adenosine levels during coronary occlusion.

Ischemic preconditioning has been proposed to protect the heart against infarction by increasing 5'-nucleotidase (5'-NT) activities and augmenting adenosine levels during sustained coronary artery occlusion. To test this theory, anesthetized dogs received four 5-min episodes of preconditioning ischemia, pretreatment with the pharmacological "preconditioning mimetic" monophosphoryl lipid A (MLA, 35 micrograms/kg i.v.) or no intervention before coronary artery ligation. At 20 min into occlusion (the crucial time at which myocyte death begins in this model), myocardial samples were obtained for measurement (by high-performance liquid chromatography) of ectosolic and cytosolic 5'-NT activity and adenosine levels. Preconditioning and MLA pretreatment limit infarct size in the canine model by 75 and 50%, respectively. However, only MLA augmented 5'-NT activity [i.e., cytosolic 5'-NT in the ischemic subendocardium was 26 +/- 1, 39 +/- 7, and 26 +/- 6 nmol. mg protein-1. min-1 in preconditioned, MLA, and control groups (P < 0.05), respectively]. Moreover, adenosine levels (in nmol/mg protein) were increased with MLA treatment (2.30 +/- 0.44) but attenuated in preconditioned dogs (1.11 +/- 0.23; P < 0.05) versus controls (1.87 +/- 0.29). Thus 5'-NT and adenosine levels need not be increased beyond control values during sustained occlusion to elicit cardioprotection.

Role of inducible nitric oxide synthase in pharmacological "preconditioning" with monophosphoryl lipid A.

Pretreatment with monophosphoryl lipid A (MLA) can pharmacologically mimic the second window of ischemic preconditioning (SWOP) to protect the heart from prolonged ischemia and reperfusion injury. Based on the delayed time course for development of MLA associated cardioprotection, this study was designed to test if MLA's cardioprotective effect is mediated by signalling through production of inducible nitric oxide synthase (iNOS), a proposed effector of SWOP. Rabbits were assigned to one of four groups: (1) vehicle control; (2) MLA: (3) vehicle+aminoguanidine (AMG) control; or (4) MLA+AMG. Monophosphoryl lipid A (35 micrograms/kg) or vehicle was given intravenously 24 h before ischemia. The selective iNOS inhibitor AMG (300 mg/ kg) was injected subcutaneously 1 h before ischemia. All rabbits experienced 30 min coronary artery occlusion followed by 3 h of reperfusion. Infarct size was measured by triphenyltetrazolium chloride (TTC) staining. followed by 3 h of reperfusion. Infarct size was measured by triphenyltetrazolium chloride (TTC) staining. Myeloperoxidase activity, an index of neutrophil infiltration, was also quantified in heart tissue collected from the post-ischemic viable border zone surrounding the infarct area. MLA pretreatment significantly reduced infarct size and neutrophil infiltration in rabbit hearts compared to control (P < 0.05). Inhibition of iNOS activity by AMG abolished the infarct size reductive effect of MLA. Aminoguanidine also blocked the ability of MLA to significantly reduce neutrophil infiltration. Although measurement of iNOS activity did not show induction of the enzyme in normal myocardial tissue 24 h after MLA pretreatment, an increase in iNOS activity in ischemic tissue relative to non-ischemic tissue was found after either 15 or 30 min of coronary occlusion in MLA treated rabbits. These results suggest that MLA pretreatment may enhance iNOS enzyme activity by MLA during ischemia which may be responsible for the observed cardioprotection.

KATP channels mediate late preconditioning against infarction produced by monophosphoryl lipid A.

The cardioprotective effect of myocardial preconditioning (PC) to reduce infarct size has been shown to last approximately 90 min (early PC), and then a second window of protection (SWOP or late PC) appears 24 h later. Although much work has been done to characterize early PC, little has been done to investigate potential mediators of SWOP. To that end, we have used monophosphoryl lipid A (MLA), a nontoxic endotoxin derivative, to produce SWOP and have examined the role of ATP-sensitive potassium (KATP) channels in mediating its cardioprotection. Adult mongrel dogs were given MLA (3, 10, or 35 micrograms/kg i.v.) 24 h before a 60-min left anterior descending coronary artery occlusion and 3 h of reperfusion. After reperfusion, the hearts were stained for myocardial infarction with triphenyltetrazolium. MLA produced a dose-dependent reduction in infarct size that was associated with an enhanced shortening of the monophasic action potential duration during early ischemia. To further examine the role of KATP channels, animals were treated with MLA (35 micrograms/kg) and 24 h later were administered either glibenclamide (0.3 mg/kg i.v.) or 5-hydroxydecanoate (7.5 mg/kg intracoronary over 20 min), two structurally distinct KATP-channel antagonists. Both glibenclamide and 5-hydroxydecanoate abolished the cardioprotection produced by MLA. These results demonstrate that the cardioprotective effect of late PC produced by MLA is dependent on functional KATP channels and is the first study to suggest that late PC may be the result of an increased KATP current during ischemia.

Myocardial ischemia/reperfusion protection using monophosphoryl lipid A is abrogated by the ATP-sensitive potassium channel blocker, glibenclamide.

OBJECTIVES: Monophosphoryl lipid A (MLA), a detoxified derivative of the lipid A portion of the endotoxin molecule, given as a pretreatment 24 h prior to cardiac ischemia/reperfusion reduces myocardial stunning and infarction in dogs. This study was undertaken to evaluate the ability of MLA pretreatment to reduce infarct size in a rabbit model of in situ regional myocardial ischemia and reperfusion. Secondly, the potential role of modulation of ATP-sensitive potassium (KATP) channel in MLA's cardioprotection was evaluated using in vivo pharmacologic antagonism with a KATP channel blocker, as was the role of tumor necrosis factor using an enzyme-linked immunosorbent assay method of serum cytokine analysis. METHODS: Rabbits were pretreated intravenously with MLA or vehicle injection 24 h prior to initiation of 30 min in situ left anterior descending coronary artery occlusion followed by 3 h reperfusion. In animals receiving glibenclamide, the potassium channel antagonist was administered 30 min prior to inducing ischemia. Animals receiving glibenclamide, which possesses hypoglycemic effects, underwent serial blood glucose evaluation prior to drug and throughout the ischemia and reperfusion periods. Hemodynamics were monitored; infarct size and area at risk were assessed by contrast dye staining (triphenyltetrazolium chloride). Serum tumor necrosis factor was measured by enzyme-linked immunosorbent method in animals administered cardioprotective doses of MLA as well as pyrogenic doses of MLA and endotoxin (positive control) to determine if elaboration of this cytokine could be associated with the cardioprotective effect of MLA. RESULTS: MLA administered as a single intravenous dose 24 h prior to ischemia reduced infarct size, expressed as a percent of the area at risk, 64 and 71% at doses of 35 and 10 micrograms/kg, respectively. Lower doses of MLA (2.5 and 5 micrograms/kg) did not significantly reduce infarct size. Administration of glibenclamide (300 micrograms/kg) 30 min prior to ischemia completely blocked the ability of MLA pretreatment to limit infarct size, while MLA vehicle-glibenclamide-treated control rabbits displayed infarcts not significantly different from MLA-vehicle-treated control rabbits. A cardioprotective dose of MLA (35 micrograms/kg) did not induce the elaboration of tumor necrosis factor into rabbit serum (within the limits of assay sensitivity). CONCLUSIONS: Single-dose pretreatment with MLA administered intravenously to rabbits substantially reduces infarct size when administered 24 h prior to ischemia. Pharmacologic preconditioning with MLA appears to be mediated through KATP channels as the channel blocker, glibenclamide, reversed the cardioprotective activity of MLA when administered 1 day following MLA pretreatment, yet 30 min prior to ischemia. In this model the cardioprotective does not appear to be associated with increases in serum tumor necrosis factor.

Cardioprotection with ischemic preconditioning and MLA: role of adenosine-regulating enzymes?

Both ischemic preconditioning and pretreatment with the endotoxin derivative monophosphoryl lipid A (MLA) protect the heart against infarction, yet the cellular mechanisms responsible for the cardioprotection achieved with either intervention are unknown. Using pentobarbital-anesthetized dogs, we tested the hypothesis that increased activity of 5'-nucleotidase (5'-NT), the enzyme that catalyzes the formation of adenosine from AMP, may play a role. Twenty-two dogs underwent 1 h of coronary occlusion and 4 h of reperfusion: eight controls received no intervention, seven animals were preconditioned with four 5-min episodes of brief ischemia, and seven received MLA (35 micrograms/kg iv) 24 h previously. Collateral blood flow was measured by injection of radiolabeled microspheres, infarct size was delineated by tetrazolium staining, and myocardial 5'-NT activities were measured by quantifying the release of adenosine from AMP. Despite comparable values of collateral blood flow in all groups, infarct size was reduced in preconditioned and MLA-treated dogs vs. controls. In addition, 5'-NT activities were increased throughout the heart with preconditioning and MLA treatment. However, single and multivariate regression analyses revealed no correlation between infarct size and 5'-NT activities for either treatment group. In fact, in the preconditioned cohort, animals with the highest enzyme activities developed the largest infarcts. This dissociation between infarct size and 5'-NT suggests that increased activity of 5'-NT is not the mechanism by which preconditioning or MLA treatment protects the canine heart against infarction.

Pharmacologic myocardial preconditioning with monophosphoryl lipid A (MLA) reduces infarct size and stunning in dogs and rabbits.

As the mechanism of ischemic preconditioning unfolds, various strategies for inducing pharmacologic preconditioning become apparent. Adenosine receptor agonists, KATP channel activators, and endothelial-neutrophil adhesion antagonists have enjoyed cardioprotective activity against ischemia/reperfusion injury in at least some preclinical models. Monophosphoryl lipid A (MLA), a structural derivative of the pharmacophore of endotoxin, enjoys an improved therapeutic index in relation to the parent biological product. MLA has found clinical application as a vaccine adjuvant and protects from sepsis and septic shock in the preclinical setting. In animal models of myocardial ischemia/reperfusion injury, pretreatment 12-24 hours prior to ischemia with a single IV bolus injection of MLA limits infarct size 50 to 75 percent in standard canine and rabbit models at doses of 10-35 micrograms/kg. Regional myocardial stunning following multiple 5-minute ischemic episodes as assessed by segment shortening is reduced in dogs pretreated 24 but not 1 hour prior to ischemia. Global cardiac function, as evaluated by pressure-volume constructs generated in dogs being weaned from cardiopulmonary bypass, recovers more quickly in animals pretreated with MLA. Cardiac protection in various models is associated with preservation of ATP during ischemia, induction of 5' nucleotidase and enhancement of calcium reuptake by SR during reperfusion. Limitation of infarct size by MLA in dogs and rabbits can be reversed by the administration of glibenclamide just prior to ischemia, suggesting a role for KATP channel opening during the first minutes of sustained ischemia. A clinical formulation of MLA (MPL-C) is currently undergoing clinical investigation in the Phase II setting in coronary artery bypass surgical patients. MLA may represent a novel means of inducing pharmacologic preconditioning, with potential for clinical application as a pretreatment before planned myocardial ischemia.