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Ann Thorac Surg 1999;68:1954-1959
© 1999 The Society of Thoracic Surgeons
a Department of Surgery, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, USA
b Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, USA
c Department of Emergency Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, USA
Address reprint requests to Dr Abd-Elfattah, Department of Surgery, Medical College of Virginia, Virginia Commonwealth University, PO Box 980532-MCV, Richmond, VA 23298-0532
e-mail: anwar{at}hsc.ucu.edu
Presented at the International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, Sep 21–24, 1997.
Abstract
Background. Monophosphoryl lipid-A (MLA) has a late window (24 hours) of cardioprotection against acute myocardial infarction. It is not known whether MLA, administered, 24 hours before surgery, attenuates intraoperative ventricular dysfunction "stunning" associated with aortic cross-clamping and reperfusion during elective cardiac surgery. We determined the dose-response relationship between MLA and ventricular function in a canine model of global myocardial stunning in the absence of necrosis. The role of expression of inducible heat shock protein 70 (HSP 70i) was also investigated.
Methods. Mongrel dogs (n = 32) were intravenously injected with either a vehicle solution or 3, 5, 10, 35 ug/kg MLA. Twenty four hours later, dogs were anesthetized and instrumented, in situ, to monitor the left ventricular performance (the slope of regression between stroke-work and end diastolic length). Tissue samples were obtained to determine HSP70i using immunoblot analysis. After a period of equilibration on cardiopulmonary bypass, the aortic cross-clamp was applied at normothermia for 30 minutes followed by 60 minutes of reperfusion. ATP and catabolites were determined in transmural myocardial biopsies. Triphenyl-tetrazolium chloride (TTC) staining was used to determine myocardial necrosis.
Results. MLA treatment did not alter myocardial contractility or ATP metabolism. Global ischemia resulted in about 50% depletion of ATP and remained depressed during reperfusion in all groups. MLA-treated hearts had improved functional recovery in a dose dependent-manner. Significant recovery was observed at the highest dose (35 ug/kg) compared to the control group. Immunoblot analysis demonstrated significant increase in HSP 70i in the MLA-treated hearts.
Conclusions. MLA exhibits a delayed (24 hours) window of protection against myocardial stunning associated with aortic cross-clamping. HSP70i expression may play a role in MLA-mediated cardioprotection.
Monophosphoryl lipid-A (MLA), a relatively nonpyrogenic derivative of endotoxin [1], activates macrophage–monocyte cell lines in a fashion similar to that of endotoxin [2]. Administration of endotoxin or MLA, 24 hours before global ischemia, attenuated myocardial dysfunction in rat hearts [3, 4]. Similarly, 24 hours after treatment, MLA protected against myocardial stunning and infarction induced by coronary artery occlusion in dogs [5–8]. MLA protected the myocardium in a rabbit model of cardiogenic shock when given 24 hours prior to ischemia, but was not protective when administered only during ischemia [9]. A delayed window of preconditioning against myocardial infarction has been observed 24 hours after exposure to a brief period of heat shock [10], ischemia [11], or administration of the A1 receptor agonists [12]. It is not known whether MLA provides a delayed window of pharmacologic preconditioning against global stunning during open heart surgery. Therefore, this study was designed to determine the efficacy and establish the dose-response relationship between of MLA and recovery of ventricular function in a surgical animal model of global ischemia while on bypass. We also determined whether MLA protection is associated with expression of heat shock protein 70i (HSP70i) in a dose-dependent manner.
Material and methods
The following studies conform to the guiding principles of the American Physiological Society. All animals were treated humanely in accordance with the United States Public Health Service Standards as outlined in "Principles of Laboratory Animal Care" formulated by the National Society of Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health. All surgical protocols have been approved by the Institutional Animal Care and Use Committee in the Medical College of Virginia, Virginia Commonwealth University.
Biochemical reagents were purchased from Sigma Chemical Company (St. Louis, MO). MLA was received from Ribi ImmunoChem, Inc (Hamilton, MT).
Thirty-two microfilaria-free adult dogs of either sex weighing 19 to 24 kg were anesthetized with sodium pentobarbital, 35 mg/kg, as an initial intravenous injection followed by 10 mg/kg when needed (Veterinary Labs, Inc, Lenexa, KA). Dogs were intubated and mechanically ventilated using a Bennett MA1 respirator (Puritan, Berkeley, CA). The azygoos vein was ligated, and the right phrenic nerve was transected to eliminate diaphragmatic contractions. The sinoatrial node was crushed, and the right atrium was paced at 150 beats per minute using a Medtronic 5880A pacemaker (Minneapolis, MN). Porcine-based heparin was injected intravenously 400 U/kg as an initial injection followed by 200 U/kg/h (Elkins-Sinn Inc, Cherry Hill, NJ). Cardiopulmonary bypass was established employing subclavian artery cannulation and atrial venous cannulation using a two-stage cannula. A membrane oxygenator (Medtronics) was used and primed with noncrossmatched homologous blood (1 dog for each experiment, n = 32). The mean reperfusion pressure was maintained at 60 to 65 mm Hg. Arterial blood gases, pH and hematocrit were routinely determined and maintained at the following levels: pO2 = 100 to 140 torr, pCO2 = 30 to 40 torr, pH = 7.32 to 7.48, and hematocrit at about 30%.
Assessment of left ventricular performance
Left ventricular performance was assessed from the relationship between stroke work and end-diastolic dimension as a sensitive and load-independent index of contractility [13]. Briefly, all pressure measurements were obtained utilizing intraventricular micromanometer-tipped catheters (Millar Instruments, Houston, TX). Left ventricular dimension data were obtained using pulse transit sonomicrometry (Triton Technology, San Diego, CA). One pair of LTZ-piezoelectric hemispheric crystals (Channel Industries, Santa Barbara, CA) were sutured to the anterior and posterior aspects of the epicardial surface of the left ventricle wall in the plane of the minor axis. The spacing between the minor axis crystals ranged from 40 to 66 mm. Analog data were digitized into a 486 PC. Subsequent analysis was performed using interactive software developed in our laboratory. Several parameters such as heart rate, systolic and diastolic left ventricular and arterial pressure, positive and negative first derivatives of pressure, ventricular dimensions and work loops are simultaneously monitored on screens and recorded on magnetic disks. In order to create work loops, the venous line and left ventricle vent were clamped, and the left ventricle and systemic pressure were allowed to rise up to 100 to 120 mm Hg using the bypass roller pump followed by separating the animal from bypass. Left ventricular blood volume was gradually removed from the heart, thus generating a family of progressively diminishing pressure-dimension work loops. The slope of the integrated work loops plotted against end-diastolic length were calculated. It has been well established that a load-independent index of contractility represents an accurate measurement of ventricular function.
Assessment of adenine nucleotide pool metabolism
Transmural Serial Tru-Cut needle (Travenol Laboratories, Inc, Deerfield, IL) biopsy specimens (5 to 10 mg) were obtained prior to ischemia and after 30 minutes of normothermic ischemia, and after 30 and 60 minutes of reperfusion. Biopsies were immediately frozen and stored in liquid nitrogen. Each biopsy specimen was extracted in 12% trichloroacetic acid (4°C) for 30 minutes with frequent homogenization. The soluble acid extract was separate from denatured protein by centrifugation and neutralized with 2:1 (v/v) of tri-n-octylamine/freon mixture (1:3 v/v) while the protein in the pellet was determined as previously described [14]. The neutralized extracts were stored at -70°C until analysis. Myocardial adenine nucleotide pool intermediates were eluted and quantified using high performance liquid chromatography using external standards [15].
Expression of heat shock protein
Myocardial tissue samples, frozen in liquid nitrogen, were thawed and homogenized in 10 mmol/L sodium phosphate buffer containing 1% mercaptoethanol, 5% sodium dedocylsulfate (SDS), and 10 µmol/L p-methyphenylsulfonylfloride using a polytron tissue homogenizer. The homogenate was then centrifuged for 5 minutes at 14,000 g. The supernatant fraction was separated and aliquotes were obtained to measure protein concentration with the remainder of the sample divided into smaller volume and immediately frozen in liquid nitrogen. Protein concentration was determined by the Bio-Rad Protein assay using bovine serum albumin as a standard. Samples were loaded on a 12.5% polyacrylamide gel (1 to 2 mm thick) and separated by the dissociating SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli [16]. Samples with equal protein concentrations were loaded on the gel. After SDS-PAGE, standards and sample protein bands were transferred by electroelusion to Western polyvinyllidene difluoride membrane (Schleicher and Schuell, Keene, NH) and then blocked with nonfat dry milk. Membranes were incubated with a mouse monoclonal antibody (1:1,000 dilution) that cross-react with heat shock protein 70 (Stressgen Biotechnologies Corp, Victoria, BC, Canada). The membrane was then incubated with the second rabbit antimouse IgG labeled with horseradish peroxidase (1:1,000 dilution). The membrane was developed with enhanced chemoluminscence (Amersham, Arlington Heights, IL) and exposed to x-ray film for an appropriate time period.
Protocol
Dogs were randomly assigned to 1 of 4 groups: the control group (n = 8) receiving intravenous injection of the vehicle solution and 3 MLA-treated groups (n = 8 each) where dogs were intravenously injected with 3.5, 10, or 35 µg/kg, respectively. The vehicle solution contained 10% ethanol, 40% propylene glycol in water for injection; MLA solution (300 µg/mL in the vehicle). Twenty-four hours after injection, animals were anesthetized and prepared for cardiopulmonary bypass and instrumentation. Control dogs received a corresponding volume of injection vehicle (0.117 mL/kg). At the beginning of each experiment, a tissue sample was obtained from the epicardium and frozen in liquid nitrogen for heat shock protein determinations. The heart was sliced into cross sections, examined and stained for infarction using 1% triphenyl-tetrazolium chloride (TTC) in sodium phosphate buffer, pH 7.4, for 30 minutes at 37°C, and then fixed in buffered formalin (4%) solution.
Statistical analysis
Data are presented as mean ± SEM. Sequential measurements were compared using repeated measures analysis of variance (ANOVA) using SAS (Statistical Analysis Software System Institute, Cary, NC). Subsequent comparisons were made between groups at each time period of the experiment by the method of Neuman and Keuls. Differences were considered significant if the probability value for comparison of least square means was less than 0.05.
Results
Adenine nucelotides and nucleosides
Administration of different doses of MLA or vehicle did not change baseline left ventricular performance or myocardial adenosine triphosphate (ATP) and metabolite levels 24 hours later. Myocardial adenine nucleotides levels (ATP, adenosine diphosphate [ADP], and adenosine monophosphate [AMP]) were similar at baseline in control and MLA-treated hearts. Thirty minutes of global normothermic myocardial ischemia reduced myocardial ATP by about 50% in all groups (data not shown). Myocardial ATP levels remained depressed after ischemia and did not improve during reperfusion. Overall, there were no significant differences between the MLA treated groups at different doses and the control groups with respect to ATP depletion or recovery.
Myocardial levels of both ADP and AMP were transiently increased during ischemia in the control and MLA-treated groups but decreased toward baseline during reperfusion. There were no significant differences between treatment groups with respect to ADP and AMP accumulation and utilization. Myocardial adenosine and inosine levels were significantly increased during normothermic ischemia but returned to basal levels during reperfusion and did not differ from MLA pretreatment. Myocardial inosine levels were about 10-fold at the end of 30 minutes ischemia. Accumulation and disappearance of myocardium hypoxanthine and xanthine were not significantly different between treatment groups during ischemia and reperfusion. Similarly, myocardial NAD+ levels were not different in all groups during ischemia and reperfusion (data not shown).
Ventricular performance
Hemodynamics parameters such as LV peak systolic pressure, end systolic pressure, positive and negative (systolic and diastolic) dp/dt, systolic, diastolic, and mean pressures were not significantly different at baseline between the MLA-treated and untreated groups. However, hemodynamic parameters were reduced after ischemia in the control and unrecovered. LV performance was measured by using a load-independent assessment. The function of the heart was determined at variable but diminishing filling volumes, in accordance with Starling’s Law. This maneuver allows creation of progressively declining hemodynamic parameters and correspondingly diminished work loops. Left ventricular performance (SW/EDL slope) at baseline was similar in control and MLA-treated groups,
At baseline, LV performance in the control and in the groups treated with 3.5, 10, and 35 µg/kg MLA were as follows (in dyn/cm2 x 103) 83.45 ± 12.3, 99.88 + 10.9, 87.73 + 7.7, and 103.41 ± 13.6, respectively, with an average of 93.49 + 4.81 dyn/cm2 x 103 (Fig 1). Persistent myocardial dysfunction was observed in the untreated controls at 30, 60, 90, and 120 minutes of reperfusion, 29.83 + 10.9, 42.40 + 22.3, 34.00 + 16.4, and 51.18 + 3.72 dyn/cm2 x 103 (p < 0.5 vs baseline 83.45 + 12.3 dyn/cm2 x 103). However, a significant improvement of LV performance was observed in the group treated with 35.0 µg/kg MLA after 30 minutes and at the end of reperfusion period. A dose-dependent relationship between the total recovery of myocardial contractility and MLA doses was observed during reperfusion. The percent recovery of LV function was 47.19 + 5.69, 60.10 + 5.8, 64.66 + 3.12, and 70.86 + 5.96 in the control and in the groups treated with 3.5, 10, 35 µg/kg, respectively. Overall, there were no significant differences between control and MLA-treated group in their behavior toward ischemic and reperfusion injury (ANOVA, p = NS). However, there were significant differences between groups at 30 and 90 minutes of reperfusion (modified t-test with repeated measures). Upon releasing the cross-clamp, all hearts experienced ventricular fibrillation regardless of pretreatment and were successfully defibrillated with a single electric shock (15 joules) (p = NS between groups).
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Expression of heat shock protein
Immunoblot analysis and densitometry revealed that MLA-treatment resulted in significantly higher expression of the inducible HSP 70 at all dose levels tested (Fig 2). There was no correlation between the amount of HSP70i expressed and MLA doses. The increase in expression of inducible HSP 70 in MLA-treated groups was about sixfold higher than in the control and the vehicle group, respectively.
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The rationale of the present study was to establish the dose-response relationship between MLA and recovery of ventricular function after reversible injury "myocardial stunning" induced by aortic cross-clamping and reperfusion while on cardiopulmonary bypass. Animals that received MLA 24 hours before surgery, demonstrated normal hemodynamics before ischemia and better recovery of ventricular function after unprotected normothermic ischemia and reperfusion. These observations suggest that MLA is a safe agent that protects the myocardium against injury-sustained aortic cross-clamping and reperfusion. The mechanism by which MLA provides cardioprotection in this model is not known. However, the late nature of efficacy of this drug suggest that MLA may activate endogenous mechanisms of protection that preconditions the myocardium against contractile dysfunction. These mechanisms may involve transcriptional and translational activities and synthesis of cardioprotective factors. Indeed, results from the present study demonstrated that MLA-pretreatment activated expression of the HSP70i 1 day after treatment. Whether this molecular response to MLA treatment is a major mechanism of cardioprotection is not known from the present study. Therefore, MLA may be beneficial when administered 24 hours before elective cardiac surgery.
Although MLA improved recovery of ventricular function, it did not prevent ATP depletion during ischemia or preferentially cause accumulation of the cardioprotective nucleoside, adenosine. The mechanisms by which MLA improves the outcome of the heart after global ischemia on bypass are not known. It has been reported that MLA pretreatment reduces neutrophil infiltration into postischemic viable myocardium in canine [7] and rabbit infarct models [9]. Pretreatment of leukocytes with MLA also reduces integrin mediated binding to cytokine activated vascular endothelium [17]. The ability of this drug to modulate cellular inflammatory responses [18] may play a role in reducing myocardial ischemic injury during surgery. However, there are no data available to support this speculation. MLA preconditions the dog heart against infarction [19]. As a result of ischemic or oxidative stress, the myocardium can undergo adaptive changes which reduce contractile efficiency and, hence, metabolic demand during and following sustained stress challenge. This adaptive response makes the heart less vulnerable to further stress.
It has been shown that the parent compound of MLA, endotoxin, causes toxic shock and death in animals and humans [20]. It modulates the immune system function and activates several endogenous mechanisms, which paradoxically induces tolerance to endotoxic shock when administered at low doses prior to high dose of endotoxin challenge [21]. Also, free radicals have been implicated into endotoxin-induced tolerance to further challenge. Whether MLA also induces free radicals after administration is not known from the current study.
Classical ischemic preconditioning against infarction is induced by brief ischemic periods [22]. This protection disappears within 1 to 2 hours and then reappears 12 to 24 hours later. Delayed precondition has also been demonstrated following ischemia [23], heat stress [24], or treatment with an adenosine receptor agonist such as 8-chloro-N-6-cyclopently-adenosine (CCPA) [12]. Since MLA did not augment endogenous adenosine during ischemia, the delayed window of protection may not be mediated by an activated A1 receptor. Adenosine triphosphate sensitive potassium (KATP) channels have been shown to open in response to ischemia and are thought by many to be the end effector of the first window of preconditioning associated with ischemic or adenosine mediated preconditioning [25–27]. Several reports demonstrated the important role of KATP channels in MLA-induced delayed preconditioning [26, 27]. KATP antagonists such as glibenclamide and 5-hydroxydeconate block MLA induced limitation of infarction in canine [27] and rabbit models of acute myocardial infarction [28].
The mechanisms by which MLA attenuated global myocardial stunning in the present study also remain to be fully elucidated [29]. However, it is plausible that MLA may have activated certain endogenous mechanisms of protection that probably preconditioned the myocardium within 24 hours against anticipated contractile or vascular injury associated with warm global ischemia. The cardioprotective effects of MLA seems to resemble that of the delayed preconditioning phenomenon which has been observed 24 hours following transient ischemia or heat shock. MLA-mediated cardioprotection concurs with a molecular response and a significant expression of the HSP70i. The correlation between MLA doses and contractile recovery and expression of HSP70i could not be demonstrated in the present study. It is postulated that expression of the inducible HSP70 may contribute, in part, to MLA-mediated protection.
MLA mediated protection against infarction is time-dependent and peaks between 6 and 24 hours following drug administration [29]. However, MLA does not protect against infarction when administered during ischemia [9] or earlier than 4 to 6 hours prior to ischemia, suggesting that time may be required for MLA-induced transcriptional and transitional expression of endogenous cardioprotective factors. Since maximal protection is commonly observed 24 hours after drug administration, it could be suggested that MLA exerts its protective effects following synthesis and/or enhanced expression of potentially cardioprotective factors, such as heat shock proteins, manganous superoxide dismutase, or inducible nitric oxide synthase. A recent study reported that MLA reduced infarct size in a rabbit model of acute coronary artery occlusion and reperfusion but it did not induce expression of HSP70i [30]. Baxter and coworkers [31] reported strong expression of HSP 70i in the myocardial tissue in both the MLA- and vehicle-treated animals with no significant differences between groups. Other investigators have demonstrated expression of HSP 70 in endotoxin-treated animals compared to the untreated control group [32, 33]. In the present studies, HSP70i was significantly expressed sixfold greater than the vehicle and the control groups. These results confirm our recent findings that MLA induces significant expression of HSP 70i in vivo in rabbits and in vitro in an cultured cardiomyocytes and C2C12 cell line [34].
It is concluded that MLA attenuation of global myocardial stunning may be related, in part, to induction of HSP70i. MLA may have favorable benefits in events of elective surgical coronary artery bypass grafting, and possibly in valve reconstruction and replacement, and heart transplant.
Acknowledgments
This study was supported, in part, by Ribi ImmunoChem Research, Inc, and National Institute of Health Award HL 5-9010 (Anwar S. A. Abd-Elfattah).
References
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