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J Thorac Cardiovasc Surg 2004;128:170-179
© 2004 The American Association for Thoracic Surgery
Cardiopulmonary support and physiology |
a Division of Cardiothoracic Surgery, Department of Surgery, The University of Washington, Seattle, Wash, USA
b Department of Thoracic and Cardiovascular Surgery, Mie University, Tsu, Japan
Received for publication June 17, 2003; revisions received October 17, 2003; revisions received November 5, 2003; accepted for publication December 2, 2003.
* Address for reprints: Timothy H. Pohlman, MD, Department of Surgery, Division of Cardiothoracic Surgery, University of Washington, Box 356310, 1959 NE Pacific St, Seattle, WA 98195, USA
tpohlman{at}u.washington.edu
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Abstract |
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METHODS: Wild-type mice (C3H/HeN) or toll-like receptor 4 mutant mice (C3H/HeJ) were subjected to 60 minutes of regional myocardial ischemia followed by 2 hours of reperfusion. At the end of reperfusion, the area at risk and the myocardial infarct size were measured as the end point of myocardial ischemia/reperfusion injury. Myocardial mitogen-activated protein kinase activation was measured by Western blotting, and nuclear translocation of nuclear factor-
B and activator protein-1 was determined by electrophoretic mobility shift assay. Ischemia/reperfusion–injured myocardium was also assessed by ribonuclease protection assay for expression of inflammatory mediators (tumor necrosis factor-
, interleukin-1ß, monocyte chemotactic factor-1, and interleukin-6).
RESULTS: The area at risk was similar for all groups after myocardial ischemia/reperfusion injury. There was a 40% reduction in infarct size (as a percentage of the area at risk) in C3H/HeJ mice compared with C3H/HeN mice (P = .001). Within the myocardium, significant activation of c-Jun N-terminal kinase, p38, and extracellular signal-regulated kinase was observed in both strains after ischemia and during reperfusion as compared with an absence of mitogen-activated protein kinase activation during sham operations; however, c-Jun N-terminal kinase activity, but not p38 or extracellular signal-regulated kinase activity, was significantly reduced in C3H/HeJ mice (P < .05). In both groups, nuclear factor-B and activator protein-1 nuclear translocation occurred in the myocardium during myocardial ischemia/reperfusion injury, but, by densitometric analysis, nuclear translocation of nuclear factor-B and activator protein-1 was significantly decreased in C3H/HeJ mice compared with C3H/HeN mice. Interleukin-1ß, monocyte chemotactic factor-1, and interleukin-6 were detectable in reperfused ischemic myocardium but were not detected in sham-operated myocardium; the expression of each of these mediators was significantly decreased in the myocardial tissue of C3H/HeJ mice when compared with expression in the control C3H/HeN mouse strain.
CONCLUSIONS: Our data suggest that toll-like receptor 4 may mediate, at least in part, myocardial ischemia/reperfusion injury. Inhibition of toll-like receptor 4 activation may be a potential therapeutic target to attenuate ischemia/reperfusion-induced tissue damage in the clinical setting.
The cytoplasmic portions of TLRs share structural and signaling similarities with the receptor for interleukin (IL)-1.12 With binding of ligand, TLR4 activates signaling pathways that lead to nuclear translocation of nuclear factor (NF)-B, which promotes the expression of several inflammatory mediators, including tumor necrosis factor (TNF)-, IL-1ß, IL-6, and IL-8.13-15 TLR4, initially demonstrated in monocytes, has since been shown to be expressed in other tissues, including the heart.16 Injured human and murine myocardium have intense TLR4 expression.15 It is interesting to note that the expression of TLR4 within the heart is mostly in cardiomyocytes—cells that lack any identified role in immunity.
TLR4 is linked to mitogen-activated protein kinase (MAPK) activation. MAPKs, such as p38 and c-Jun N-terminal kinase (JNK), are activated during myocardial ischemia/reperfusion (I/R) injury (see review17), an event that induces an acute inflammatory reaction. We therefore hypothesized that TLR4, which elicits an inflammatory response on stimulation, mediates I/R injury in the heart. To test this hypothesis, we compared myocardial I/R injury between 2 strains of mice: C3H/HeJ and C3H/HeN. C3H/HeJ is a strain of mice that contains a missense mutation in the intracellular portion of TLR4 that prevents the activation of the remainder of the downstream signaling pathways.2,3 C3H/HeN is the mouse strain used as a wild-type control. We demonstrated in this study that after 60 minutes of regional myocardial ischemia, C3H/HeJ mice have a smaller area of infarction after reperfusion than C3H/HeN mice: this suggests participation of TLR4 in myocardial I/R injury.
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Materials and methods |
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B and activator protein [AP]-1) were examined after 15 and 30 minutes of reperfusion, respectively.
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General anesthesia was achieved with intraperitoneal pentobarbital sodium (Abbott Laboratories, North Chicago, Ill; 100 mg/kg). In all groups, anesthesia was confirmed by lack of a foot withdrawal reflex. A middle cervical incision was made, and a section of PE-90 tubing was passed through the exposed trachea until the tip was 2 mm below the larynx. Mechanical ventilation (model 687 mouse ventilator; Harvard Apparatus, Holliston, Mass) was set to ensure normal physiologic parameters after serial blood gases were obtained in pilot studies. Temperature was monitored via a rectal temperature probe (YSI Series 400 temperature probe; Yellow Springs, Ohio) and maintained at 37°C with a heating lamp. A left parasternotomy was performed under the dissecting microscope (Zeiss Operating Microscope, OPMI 6-SDFC; Oberkochen, Germany) through careful division of 3 ribs in a cephalocaudal direction parallel to the sternum, and the pericardium was reflected to expose the heart. A 7-0 silk suture (Tyco Health Care, US Surgical, Norwalk, Conn) on an HE-7 needle was passed behind the left anterior descending (LAD) artery just below the left atrial appendage. A Rumel-type snare was created by passing both ends of the suture through the tip of a 22-gauge angiocatheter that could then be tightened and released by sliding a Voss clip down on the angiocatheter for ischemia and reperfusion, respectively. Effective occlusion of the LAD was visually confirmed by blanching of the left ventricle (LV) and subsequent hyperemia with release of the snare. After 60 minutes of ischemia, the occlusive snare was released to allow reperfusion of up to 2 hours (Figure 1). Sham mice underwent the same surgical procedures except that the suture was not snared. At the completion of reperfusion, hearts were rapidly explanted for molecular analyses. The LV was dissected free, rinsed in 0.9% saline, snap-frozen in liquid nitrogen, and stored at –80°C until subsequent analysis. Hearts that were analyzed for area at risk (AAR) and infarct size were harvested after 2 hours of reperfusion.
Determination of AAR and infarct size
At the completion of the experimental protocol, the LAD was reoccluded, and 4% Evans blue dye was injected into the aortic root to determine AAR (unstained area). Hearts were then explanted, rinsed in 0.9% normal saline, embedded in 1% agarose gel (UltraPure agarose; Invitrogen, Carlsbad, Calif) in phosphate-buffered saline (pH 7.4), and sliced into 1-mm-thick sections parallel to the atrioventricular groove. Each heart section was incubated in 1% triphenyltetrazolium chloride at 37°C for 10 minutes and in 10% formalin for 24 hours and was then weighed and photographed with a digital camera (Coolpix 950; Nikon, Tokyo, Japan). AAR and infarct area (unstained by triphenyltetrazolium chloride) were measured by using computer planimetry (ImageJ 1.21; National Institutes of Health public domain, http://rsb.info.nih.gov/ij), and the average values were recorded. These areas were measured in a blinded fashion by an observer unaware of the experimental groups. Infarct volumes were calculated and expressed as a percentage of AAR.
Preparation of cytosolic and nuclear proteins
Frozen-tissue samples of the LV were ground into a fine powder and homogenized in ice-cold lysis buffer. The homogenates were centrifuged at 15,000g at 4°C for 10 minutes, and the supernatants were collected as whole myocardial extracts.
For nuclear extractions, heart tissue samples were suspended in an ice-cold low-salt buffer containing 0.6% Nonidet P-40 (Sigma, St Louis, Mo). The solution was homogenized and centrifuged at 350g at 4°C for 20 seconds. The pellet was discarded, and the supernatant was centrifuged again at 15,000g at 4°C for 10 minutes. The supernatant was saved as cytosolic extract; the pellet was suspended in 40 µL of high-salt buffer* at 4°C for 20 minutes. This solution was then centrifuged at 15,000g at 4°C for 10 minutes, and the supernatant was collected as the nuclear protein extract. Final protein concentrations of these extracts were determined by using the bicinchoninic acid method (Pierce Biotechnology, Inc, Rockford, Ill). Samples were stored at –80°C until the time of assay.
Western immunoblotting assay for phosphorylation of MAPKs
Whole-cell myocardial extracts were resolved on 10% to 15% sodium dodecyl sulfate-polyacrylamide electrophoretic gels as previously described.19 Gels were transferred to polyvinylidene difluoride membranes, washed, and incubated with primary antibodies for the activated, phosphorylated forms of p38, ERK, and JNK (1:1000; Cell Signaling Technology Inc, Beverly, Mass). Blots were also reprobed with antibody for total protein (p38, ERK, or JNK). Blots were incubated with horseradish peroxidase–conjugated secondary antibody (1:2000) in blocking buffer with gentle agitation for 1 hour at room temperature. Immunoreactivity was detected with enhanced chemiluminescence (Pierce) and quantitated with densitometry (NIH Image 1.62). The ratio of phosphorylated- to total MAPK immunoreactivity was determined for each sample, and the results were expressed as fold activation over a control.
Electrophoretic mobility shift assays for NF-B and AP-1 activity
Electrophoretic mobility shift assays (EMSA) using an oligonucleotide containing the consensus sequence motifs for NF-B binding (5'-AGTTGAGGGGACTTTCCCAGGC-3') and AP-1 binding (5'CGCTTGATGAGTCAGCCGGAA-3'; Promega, Madison, Wis) were performed as previously described.20 Densitometry was performed with NIH Image 1.62. The results were expressed as fold activation over control.
Ribonuclease protection assay
Total RNA was isolated from frozen tissue samples by the guanidinium thiocyanate-phenol-chloroform method. RNA integrity was confirmed by agarose gel electrophoresis and quantitated by optical density measurements at 260 nm. RNA was evaluated with an RPA III Ribonuclease Protection Assay Kit (Ambion Inc, Austin, Tex) and a customized mouse cytokines template (Riboquant Multi-Probe Template Set; BD Biosciences Pharmingen, San Diego, Calif). In vitro transcription was performed in buffer supplemented with [32P]uridine triphosphate (PerkinElmer, Inc., Wellesley, Mass) and T7 RNA polymerase (MAXIscript T7 Kit; Ambion). Labeling efficiency was determined by measuring Chernokov activity in a scintillation counter. Each riboprobe was diluted to the optimal activity (defined by the manufacturer), added to 20 µg of heart RNA, heated for 3 minutes at 95°C, and then hybridized at 56°C overnight. After ribonuclease and proteinase K treatment, protected RNA hybrids were purified by phenol and chloroform extraction and ammonium acetate and ethanol precipitation and then separated by electrophoresis on 5% polyacrylamide/8 mol/L urea gels. Gels were dried and subjected to autoradiography. Densitometric analysis was performed with NIH Image 1.62.
Statistical analysis
All data are expressed as mean ± SEM. The significance of the difference between group means was analyzed by analysis of variance with post hoc comparisons by the Scheffé protected least significant difference test. All statistical analyses were performed with StatView 6.0 (SAS Institute Inc, Cary, NC).
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Results |
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B and AP-1)B and AP-1 was determined by using EMSA to measure nuclear translocation during myocardial I/R injury. EMSAs revealed activation of both NF-B and AP-1 in both C3H/HeJ and C3H/HeN mice. However, by densitometric analysis of the gels, with normalization of band density to total protein loaded, the apparent amounts of NF-B and AP-1 translocations were significantly reduced in C3H/HeJ mice compared with wild-type controls (C3H/HeN; P < .05; Figure 3, B). Neither NF-B activation nor AP-1 activation was observed at any point in any experiment during the reperfusion period in the sham-operated C3H/HeN mice (data not shown).
Expression of inflammatory cytokines and chemokines
The mRNA expression of TNF, IL-1, IL-6, and monocyte chemotactic factor (MCP)-1 in cytosolic fractions of homogenized LV from sham-operated wild-type C3H/HeN and TLR4 mutant C3H/HeJ mice were examined after 60 minutes of ischemia and 120 minutes of reperfusion for each strain (Figure 4). Myocardial I/R in C3H/HeN mice (n = 4) induced upregulation of IL-1, IL-6, and, most prominently, MCP-1 when compared with sham controls, in which accumulation of mRNA for IL-1, MCP-1, and IL-6 was only barely detectable. However, IL-1, MCP-1, and IL-6 were not detected in C3H/HeJ mice during myocardial I/R injury (n = 4). As shown in Figure 4, a small amount of TNF was induced in one wild-type mouse heart after myocardial I/R injury, but not in the other 3 experiments and in none of the 4 experiments with C3H/HeJ mice. Quantification of mRNA levels by densitometry revealed that the differences in expression of IL-1, MCP-1, and IL-6 between C3H/HeN and C3H/HeJ strains were all significant (P < .05).
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Discussion |
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B, thus targeting inhibitor B for degradation. Degradation of IB releases NF-B, which translocates into the nucleus, where it promotes the transcription of a wide variety of genes that encode proteins generally involved in the evolution of an inflammatory response or proteins that inhibit apoptotic cell death.
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Although C3H/HeJ mice are more susceptible to infection with gram-negative bacteria, TLR4 blockade has been examined as a treatment for endotoxemia.24-26 E5564 is a structural analog of the lipid A portion of LPS and is a potent antagonist of the biochemical and physiologic effects of LPS in several in vitro and in vivo models. E5564 is currently under clinical development as a possible therapeutic agent for the treatment of sepsis.26 Whether E5564 can block binding of non-LPS structures to TLR4 in myocardial I/R injury remains to be determined.
Discussion
Dr Robert C. Robbins (Stanford, Calif). I had never heard of TLR4 before getting this abstract from the program committee, so I appreciate the opportunity to be educated in this area. Dr Chong has done a very nice job in his opening remarks to discuss toll-like receptors, which are recently discovered transmembrane receptor proteins that play a central role both in the inflammatory process and in the innate and inductive immunity process. As he pointed out, most of the studies that I could find in the literature are based on septic shock, but the objective of his study was to try to better understand the involvement of TLR4 in I/R injury in the myocardium.
He used wild-type animals and then a transgenic animal that reduced the capability of toll-like receptor function, and he observed that toll-like receptor mutant mice had significantly less myocardial injury as manifested by reduction in myocardial infarct size, cell signaling proteins, transcription factor, and cytokines that are associated with I/R injury than did the control animals.
To comment technically about this study, I will just say that this work was done by beating heart ligation of the LAD in 25-g mice. These mice are about the size of the end of my finger and 10 times smaller than most of the rats that we normally operate on. Technically it is a tour de force to even accomplish this model.
Second, the molecular biology is just another reflection of Dr Verrier's incredible laboratory and the infrastructure he has. The information was well presented and the quality of the gels was outstanding. I was able to find only 2 articles in the surgical literature concerning TLR4. One is a recent article from the University of Toronto that demonstrated that the oxidative stress of hemorrhagic shock contributed to regulation of TLR4 mRNA expression, and then Magdi Yacoub's group has demonstrated that TLR4 expression was significantly increased in patients requiring left ventricular assist device support compared with stable patients with advanced heart failure who received cardiac transplantation. However, the current study is novel and significant because it is the first report, as Dr Chong has indicated, of the importance of toll-like receptor in myocardial I/R injury. As the authors correctly indicate, this new knowledge may lead to potential therapeutic strategies for the reduction of myocardial injury associated with reperfusion injury.
Dr Chong, I have just a few brief comments and questions. Do you have any comment about future plans or why you did not do any echocardiograms or dp/dt in these mice?
Dr Chong. Thank you for those kind comments, Dr Robbins. Our in vivo model used infarction size as a measure of myocardial injury. Our model is capable of producing reproducible results and allowing us to really measure what the extent of the damage from I/R injury is. The other variables such as dp/dt and also echocardiograms would only add to the data, and we are actually in the process of acquiring an ultrasound machine that would actually help us do that. Measurement of dp/dt and other hemodynamic parameters would require us to purchase other equipment to measure that in an in vivo setting. We have thought about that and we thought that the measurement of infarction size was sufficient.
Dr Robbins. I think your next level of this work is going to be to actually allow the animals to survive and look at long-term functional data. Your answer might sound like a cop-out to some people, but at Stanford there is one 15-MHz probe and we have an incredible time trying to get our animals imaged. I agree with you that it is a big expense and it is very tough to do, but I thought I would bring it up.
Have you measured the actual toll-like receptor protein expression in your animals and is it possible to create a total knock-out of this gene or would they just not survive?
Dr Chong. Another excellent question. First, the expression of TLR4 has been shown in some studies. These studies concentrate on the heart that has been damaged by ischemia chronically, and they show that the expression of TLR4 over several days and weeks is increased in the cardiomyocytes in points where the cells are juxtaposed. In our model we have not done so. It would require us to do an in situ hybridization for mRNA levels of TLR4 because the time alloted for during ischemia and reperfusion we believe is not enough to have de novo synthesis of TLR4 and to bring it up to the surface.
The second answer is, yes, it is possible to have transgenic mice with targeted deletion of TLR4. In fact, we are just about to get those mice in addition to mice that have targeted deletion of very proximal signaling molecules for all the toll-like receptors and not just TLR4. Further, we are also collaborating with several pharmacologic companies that have antagonists to TLR4 and are now proceeding with clinical studies in sepsis. Our preliminary results in our model show very promising results.
Dr Robbins. Why do you think you observed a reduction in JNK kinase but not in p38 and ERK?
Dr Chong. That's a great question. Myocardial I/R has been shown in the literature to activate all 3 classes of MAP kinases, p38, ERK, and JNK. As you know, activation of MAP kinase is very complex and it is not as simple as I have shown here. The activation of these kinases, as well as the signaling molecule targets of these kinases, varies not only on the identity of the signal but also on the duration, the strength, and the location. Because the inflammatory response in myocardial I/R is so redundant, we found that surprising to have found a reduction in JNK at all. It seems that the protection we see from inhibiting TLR4 signaling comes exclusively through JNK signaling, and we hope to investigate this further by looking at similar studies in transgenic mice with targeted deletion of TLR4 as well as MYD88 and also MAP kinase, kinase 3 and 6 deletions, which are the kinases for the MAP-kinases. Those are the ones that exclusively control JNK.
Dr Robbins. Nowhere in the literature could I find information about antagonists to toll-like receptors, so you speculated this might be useful in clinical strategies. How would you plan for us to do that? Would it be an antibody or how would we prevent toll-like receptor in a clinical setting?
Dr Chong. Cellularly, the antagonist binds TLR4 and prevents basically further downstream signaling. The first known adaptor molecule to this receptor is called MYD88, and that is crucial to the further downstream events of this receptor. Blocking it with a specific antagonist would block the recruitment of this adaptor molecule. We believe that this antagonist can be used in a variety of settings, settings in which I/R events are involved, such as organ transplantation, cardiopulmonary bypass, when you arrest the heart during cardiac surgical procedures, during revascularization procedures, and so forth.
Dr Thomas Maxey (Charlottesville, Va). Dr Chong, you and Dr Verrier again have set the mark for I/R research. As you know, the complex pathway of I/R injury involves certainly many receptors and intracellular mediators. You have beautifully demonstrated that this TLR4 down-regulates some of the intracellular things, such as p38. Did you measure any of the end products of TNF- and cellular adhesion molecules and things even more downstream from NF-B to see whether those were down-regulated as well?
Dr Chong. We measured mRNA levels of proinflammatory mediators like those you are talking about—TNF-, interleukins, adhesion molecules. As I have demonstrated in the ribonuclease protection assay, which actually is a gel of looking at mRNA levels, it was all up-regulated in the wild-type mice, and by all I mean MCP-1, IL-6, and IL-1ß. In fact, there were actually others that I did put up on the gel just for the sake of simplification, but with TLR4 mutation all these levels were decreased. Now we are planning to measure not just mRNA levels but also protein levels as well as mRNA levels by in situ hybridization within the cell so that we know which cell type these proteins come from. To do that, we have to extend our reperfusion times long enough to have de novo protein synthesis from the mRNA, and that will require longer reperfusion times. There are some technical difficulties with making the mice survive long enough for us to explant the heart.
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Footnotes |
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Read at the Twenty-ninth Annual Meeting of The Western Thoracic Surgical Association, Carlsbad, Calif, June 18-21, 2003.
* Hepes 20 mmol/L, MgCl2 1.5 mmol/L, NaCl 420 mmol/L, EDTA 0.2 mmol/L, and glycerol 25%. 
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significant decrease compared with the WT level.
