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Am J Physiol Heart Circ Physiol 292: H503-H509, 2007. First published September 15, 2006; doi:10.1152/ajpheart.00642.2006
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Toll-like receptor 2 modulates left ventricular function following ischemia-reperfusion injury

Yasushi Sakata,1,* Jian-Wen Dong,1,* Jesus G. Vallejo,1,3 Chien-Hua Huang,1,2 J. Scott Baker,3 Kevin J. Tracey,4 Osamu Tacheuchi,5 Shizuo Akira,5 and Douglas L. Mann1,2

1Department of Medicine, Winters Center for Heart Failure Research, 2Texas Heart Institute at Saint Luke's Episcopal Hospital, 3Section of Infectious Diseases, Department of Pediatrics, Baylor College of Medicine, and Texas Children's Hospital, Houston, Texas; 4Laboratory of Biomedical Science, North Shore University Hospital, New York University School of Medicine, Manhasset, New York; and 5Department of Host Defense, Osaka University, Osaka, Japan

Submitted 15 June 2006 ; accepted in final form 28 August 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Production of proinflammatory cytokines contributes to cardiac dysfunction during ischemia-reperfusion. The principal mechanism responsible for the induction of this innate stress response during periods of myocardial ischemia-reperfusion remains unknown. Toll-like receptor 2 (TLR2) is a highly conserved pattern recognition receptor that has been implicated in the innate immune response to a variety of pathogens. However, TLR2 may also mediate inflammation in response to noninfectious injury. We therefore hypothesized that TLR2 is essential for modulating myocardial inflammation and left ventricular (LV) function during ischemia-reperfusion injury. Susceptibility to myocardial ischemia-reperfusion injury following ischemia-reperfusion was determined in Langendorff-perfused hearts isolated from wild-type mice and mice deficient in TLR2 (TLR2D) and Toll interleukin receptor domain-containing adaptor protein. After ischemia-reperfusion, contractile performance was significantly impaired in hearts from wild-type mice as demonstrated by a lower recovery of LV developed pressure relative to TLR2D hearts. Creatinine kinase levels were similar in both groups after reperfusion. Contractile dysfunction in wild-type hearts was associated with elevated cardiac levels of TNF and IL-1beta. Ischemia-reperfusion-induced LV dysfunction was reversed by treatment with the recombinant TNF blocking protein etanercept. These studies show for the first time that TLR2 signaling importantly contributes to the LV dysfunction that occurs following ischemia-reperfusion. Thus disruption of TLR2-mediated signaling may be helpful to induce immediate or delayed myocardial protection from ischemia-reperfusion injury.

inflammation; innate immunity; myocardial function

REPERFUSION OF BLOOD FLOW to ischemic myocardium is in and of itself associated with a distinct form of cardiac injury that is directly attributable to the toxic effects of reactive oxygen intermediates that are generated once the heart is reperfused (6, 7, 25). Recently, it has been suggested that ischemia-reperfusion leads to increase in the expression of a portfolio of inflammatory mediators that are related to the so-called innate immune system, including TNF, IL-1beta, IL-6, and nitric oxide (9, 14, 27). This robust inflammatory response provokes a number of deleterious effects in the heart with the most notable being left ventricular (LV) dysfunction (6, 8, 9). However, an incomplete understanding of the primary mechanisms through which the heart initiates this inflammatory cascade has hampered progress in the treatment of ischemia-reperfusion-induced LV dysfunction.

Recent studies have shown that the heart possesses a functionally intact innate immune system that becomes activated nonspecifically in response to all forms of acute myocardial injury, especially during ischemia-reperfusion (22, 23). Cardiac myocytes express at least four classical receptors that belong to the innate immune system (so-called pattern recognition receptors), including CD14, and Toll-like receptors 2, 4, and 6 (TLR2, TLR4, and TLR6, respectively) (12, 13, 18, 19). TLRs are highly conserved pattern recognition receptors that have been implicated in the innate immune response to a variety of pathogens (1, 26). Recently, it has become clear that TLRs also recognize endogenous host material that is released during cellular injury or death (5). All TLRs (except for TLR3) interact with an adaptor protein termed myeloid differentiation factor 88 (MyD88) via their Toll interleukin receptor domains. When stimulated, MyD88 recruits IL-1 receptor-associated kinase to the receptor complex. IL-1 receptor-associated kinase is then activated by phosphorylation and associates with tumor necrosis receptor-associated factor 6, leading to NF-{kappa}B activation (2). Although the adaptor molecule Toll interleukin receptor domain-containing adapter protein (TIRAP) was initially thought to contribute to MyD88-independent signaling, studies have shown that TIRAP is required for TLR2- and TLR4-mediated activation of NF-{kappa}B (15, 40).

Several lines of evidence support the view that TLR2 has a broad role as a pattern recognition receptor for a variety of microbes and microbial structures. These include lipoproteins from pathogens, such as mycobacteria and staphylococcal peptidoglycan and lipoteichoic acid (11, 37, 39). We have previously shown that cardiac expression of TLR2 is essential for LV dysfunction and myocardial expression of TNF, IL-1beta, and nitric oxide following challenge with Staphylococcus aureus (19). However, TLR2 may also mediate innate stress responses following noninfectious injury (17). Indeed, a recent study in cardiac myocytes showed that hydrogen peroxide-induced oxidative stress was sufficient to increase signaling through TLR2 and that this signaling could be prevented by an anti-TLR2 antibody (12). Moreover, Leemans et al. (21) reported that renal-associated TLR2 was an important initiator of inflammatory responses leading to renal injury and dysfunction following ischemia-reperfusion. Taken together, these observations suggest that activation of TLR2 signaling during cardiac ischemic injury may be an important link between the innate inflammatory response in the heart and the LV dysfunction associated with this condition. Accordingly, in this study, we determined whether TLR2 played a role in ischemia-reperfusion-induced cardiac inflammation and LV dysfunction.


   METHODS
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 METHODS
RESULTS
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Mice. The mutant mice (129SV x C57BL/6) deficient in TLR2 (TLR2D) or TIRAP (TIRAP-D; provided by Dr. Ruslan Medzhitov, Yale University School of Medicine) expression were generated by gene targeting as described previously (15, 36). Homozygous TLR2–/– and TIRAP–/– mice were generated by intercrossing heterozygous (TLR2+/– and TIRAP+/–) mice. Wild-type littermates served as the appropriate controls. Male mice used in this study were maintained in specific pathogen-free conditions and were fed pellet food and water ad libitum. All studies were performed with the approval of the Institutional Animal Care and Use Committee at Baylor College of Medicine. These investigations conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.

Isolated heart perfusion studies. Hearts from wild-type, TLR2D, and TIRAP-D mice were isolated and perfused in the Langendorff mode as previously described (19). Briefly, mice were injected intraperitoneally with heparin (10,000 U/kg; Sigma, St. Louis, MO) and anesthetized with Avertin (16 µl/g body wt of a 2.5% solution). The thorax was rapidly opened and the heart excised and arrested in ice-cold saline. A short perfusion cannula was inserted into the aortic root for retrograde perfusion. Isolated hearts were perfused at a constant pressure of 80 mmHg with modified Krebs-Henseleit buffer containing (in mmol) 118 NaCl, 24 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.2 CaCl2, 10 glucose, and 2 pyruvate (37°C), equilibrated with 95% O2-5% CO2 to yield a pH of 7.4. The perfusate was gassed with 95% O2-5% CO2. A handmade balloon connected to a polyethylene tube was inserted into LV through the mitral valve via an incision in the left atrium and was connected to a pressure transducer (ML844, AD Instruments, Colorado Springs, CO). The balloon was inflated with water to adjust LV end-diastolic pressure (LVEDP) at 7–10 mmHg.

After a 30-min stabilization period, hearts from wild-type TLR2D and TIRAP-D mice were subjected to 30 min of zero-flow ischemia (t = –30 min) followed by reperfusion (t = 0) for 30 to 60 min (Fig. 1). All hearts were paced at 420 beats/min with pacing electrodes placed on the right atrium. Pacing was interrupted during ischemia and resumed 3 min after the start of reperfusion. Functional data were recorded at 1 kHz on a data acquisition system (PowerLab, ADInstruments). LV developed pressure (LVDP) was calculated as the difference between peak-systolic pressure and LVEDP. At the end of each experiment, hearts were frozen in liquid nitrogen for subsequent cytokine analysis.


Figure 1
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  		Fig. 1. Ischemia-reperfusion (I/R) protocol. Following stabilization, hearts were subjected to 30 min of no-flow ischemia followed by reperfusion (t = 0) for 30 to 60 min.

 
Creatinine kinase assay. Coronary effluent was collected during the first 30 min after ischemia. Creatine kinase (CK) activity was measured with a commercially available CK assay kit (Diagnostic Chemical, Charlottetown, PE, Canada) according to the manufacturer's recommendations. CK activity was normalized for frozen-dry heart weight. Data are expressed as units per gram of cardiac tissue.

TNF and IL-1beta protein measurements. Hearts were taken following the ischemia-reperfusion protocol, and homogenates were prepared as previously described (19). Commercially available ELISA kits (R&D Systems, Minneapolis, MN) were used for measuring myocardial TNF and IL-1beta protein levels. Data are expressed as picogram per milligram of protein.

TNF neutralization. In separate experiments, hearts were perfused during the period before ischemia with etanercept (30 µg/ml), a recombinant fusion protein that binds TNF and functionally inactivates it (24). This concentration of entanercept has been shown to neutralize the negative inotropic effects of TNF following ex vivo ischemia-reperfusion (10). Hearts from wild-type and TLR2D mice were then subjected to ischemia-reperfusion, and LV functional recovery was measured as described in Isolated heart perfusion studies.

Immunohistochemical staining. To localize the cellular source of high-mobility group box 1 (HMGB1) expression, we performed immunochemistry studies using a rabbit anti-HMGB1 antibody (1:1,000; BD Biosciences, San Jose, CA). Paraffin-embedded cardiac sections were used for immunostaining using an immunoenzymatic staining kit (DAKO EnVision+ Systems, Peroxidase, Dako; Carpintera, CA) as recommended by the manufacturer. Counterstaining was performed with hematoxylin, and each immunostained slide was evaluated by light microscopy.

Isolation of cytoplasmic proteins. HMGB1 released by injured or necrotic cells has been shown to act as a signaling molecule, inducing local inflammatory responses (34). Thus, extracellular HMGB1 can be regarded as both a signal of tissue injury and a mediator of inflammation. Germane to this discussion is the recent in vitro observation that TLR2 and TLR4 act as receptors for HMGB1 (31, 32). HMGB1 protein expression was assessed in the hearts of wild-type and TLR2D mice after ischemia-reperfusion. Hearts were homogenized in 2 ml of ice-cold extraction buffer as previously described (4). The protein concentration was determined by using the bicinchoninic assay with bovine serum albumin as a standard (Pierce, Life Science; Rockford, IL). Protein was separated on 12% SDS-polyacrylamide gel under denaturing conditions and was electroblotted onto a nitrocellulose membrane (Bio-Rad; Hercules, CA). The membrane was immunoblotted with rabbit anti-HMGB1 antibody (BD Biosciences). The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit polyclonal antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA). HMGB1 expression was detected with the ECL-Plus Western blotting detection kit (Amersham).

To determine whether HMGB1 was released during ischemia-reperfusion, perfusates were filtered through Millex-GP (Millipore, Bedford, MA) to clear the samples from cell debris and macromolecular complexes. Samples were then concentrated 50-fold with Amicon Ultra-4–10000 NMWL (Millipore). Western blot analysis for HMGB1 was performed as described in Isolation of cytoplasmic proteins.

Murine cardiac myocyte isolation. Isolated ventricular myocytes were prepared from wild-type and TLR2D mice with minor modifications to the method described by Rockman et al. (33). The animals were anesthetized with a mixture of ketamine (100 µg/ml) and xylazine (20 µg/ml). Hearts were excised and perfused at 2 ml/min in a Langendorff apparatus, first with heart media-3 composed of (in mM) 20 glucose, 4 NaHCO3, 12 HEPES, 30 taurine, 2 carnitine, 2 creatine, and 10 2,3-butanedione monoximine (pH 7.4) at 37°C for 5 min and then with heart media-3 containing (in mg/ml) 0.5 collagenase type B, 0.5 collagenase type D, and 0.02 protease XIV for 7–10 min. Heart media-3 was a modified commercial Joklik medium (Gibco, Carlsbad, CA). The ventricle of the digested heart was then cut into several sections and subjected to gentle agitation to dissociate the cells. Intact cells were enriched by centrifugation. Freshly isolated cell were resuspended in a buffer solution containing (in mM) 20 HEPES, 1 CaCl2, 137 NaCl, 5.4 KCl, 15 dextrose, 1.3 MgSO4, and 1.2 NaH2PO4 (pH 7.4).

Myocyte contractility. Mechanical properties of cardiomyocytes were assessed using an IonOptix MyoCam system (IonOptix, Milton, MA). In brief, myocytes were placed in a chamber mounted on the stage of an inverted microscope and superfused with a buffer containing (in mM) 20 HEPES (pH 7.4), 1 CaCl2, 137 NaCl, 5.4 KCl, 15 dextrose, 1.3 MgSO4, and 1.2 NaH2PO4. Experiments were conducted at 37°C. Cells were exposed to buffer or 1 µg/ml of recombinant human HMGB1 (rhHMGB1; 30 min). The rhHMGB1 contained 5 pg endotoxin/µg protein (Limulus assay; BioWhittaker, East Rutherford, NJ). The myocyte being studied was displayed on the computer monitor using an IonOptix MyoCam camera, which rapidly scans the image area at every 8.3 ms such that the amplitude and velocity of sarcomere shortening/relengthening was recorded with good fidelity. The cells were then field stimulated at a frequency of 0.5 Hz using a pair of platinum wires placed on the opposite sides of the chamber connected to a FHC stimulator. IonOptix Acquisition Software was used to capture changes in sarcomere length during contraction.

Statistical analysis. All data are expressed as means ± SE. Statistical significance was evaluated by two-way ANOVA. A post hoc test of protected least significant differences (Fisher) was used to determine differences among groups where appropriate. A probability value of P ≤ 0.05 was considered statistically significant.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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TLR2 deficiency improves postischemic functional recovery of isolated hearts. A control set of hearts from wild-type and TLR2D mice (n = 9) was perfused for 90 min at a constant pressure of 80 mmHg, without being subjected to ischemia. We compared systolic LV pressure, LVEDP, LVDP, and flow between the two groups. No differences were noted in the baseline physiological characteristics between wild-type and TLR2D mice (Table 1).


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  		Table 1. Baseline physiological characteristics of wildtype and TLR2D hearts

 
Hearts from wild-type and TLR2D mice were subsequently subjected to ischemia-reperfusion injury. During global zero-flow ischemia, LVEDP increased in both wild-type and TLR2D hearts. No differences were noted in LVEDP during peak contracture, the end of ischemia, or after reperfusion (data not shown). However, the recovery of postischemic contractile performance (%LVDP) was significantly greater (P ≤ 0.05) in the hearts of TLR2D mice (Fig. 2A). At 60 min after reperfusion, hearts from wild-type mice recovered 25.2 ± 1.9% of baseline LVDP, whereas hearts from TLR2D mice recovered to 33.9 ± 3.2%. These data suggest that TLR2 is involved in mediating myocardial susceptibility to ischemia-reperfusion injury.


Figure 2
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  		Fig. 2. Decreased susceptibility to myocardial I/R injury in mice deficient in Toll-like receptor 2 (TLR2D) and Toll interleukin receptor domain-containing adaptor protein (TIRAP-D) mice. A: percent recovery of left ventricular developed pressure (%LVDP) in hearts isolated from wild-type (WT; n = 9) and TLR2D (n = 9) mice during 30 min of global zero-flow ischemia and 60 min of reperfusion. Inset: depicts the creatine kinase levels in WT (n = 7) and TLR2D (n = 7) mice after myocardial I/R injury. B: %LVDP in hearts isolated from WT (n = 8) and TIRAP-D (n = 8) mice during 30 min of global zero-flow ischemia and 60 min of reperfusion. *P ≤ 0.05 vs. WT hearts.

 
CK release after reperfusion in wild-type and TLR2D mice. To determine whether decreased ischemia-reperfusion injury in TLR2D hearts was associated with differences in cardiac injury, CK release was measured in the perfusates during reperfusion of hearts from both wild-type and TLR2D mice. Total CK release from the heart was negligible in both groups before the induction of ischemia (data not shown). The levels of CK released were markedly elevated after reperfusion in both wild-type and TLR2D mice (Fig. 2A, inset); however, there was no significant difference between wild-type and TLR2D mice. Thus zero-flow ischemia resulted in similar myocyte injury in both groups. These data suggested that the measured difference in recovery of %LVDP between hearts from wild-type and TLR2D mice could not be explained simply by differences in the degree of ischemia-induced myocyte injury.

TIRAP deficiency decreases susceptibility to ischemia-reperfusion-induced LV dysfunction. Given the specificity of TIRAP for TLR2 signaling in several isolated cell types, we next investigated the effect of TIRAP deficiency on postischemic LV functional recovery (40). Figure 2B shows postischemic LV functional recovery in hearts from wild-type and TIRAP-D mice after ischemia-reperfusion. Ischemia-reperfusion resulted in significantly more LV dysfunction in wild-type hearts, as indicated by a lower recovery of LVDP relative to that in TIRAP-D hearts; that is, at 60 min after reperfusion, hearts from wild-type mice recovered 26.84 ± 1.5% of baseline LVDP, whereas hearts from TIRAP-D mice recovered to 41.8 ± 1.5% (P ≤ 0.05). These data support a role for TLR2-mediated TIRAP signaling in the pathogenesis of ischemia-reperfusion-induced LV dysfunction.

Cytokine production in the heart after ischemia-reperfusion injury. Previous studies have shown that TNF release is one of the earliest deleterious events in response to various forms of cardiac injury (16, 27). Given that TIRAP signaling has been shown to activate proinflammatory cytokines, we sought to determine whether the improvement of postischemic functional recovery in hearts from TLR2D mice was mediated through changes in cardiac TNF and IL-1beta. Tissue homogenates from nonischemic (control) and ischemic hearts were assayed by specific TNF and IL-1beta immunoassays. Figure 3, A and B, shows TNF and IL-1beta protein production in the heart at 30 and 60 min after reperfusion. Consistent with the role of TNF in myocardial ischemia-reperfusion, myocardial TNF protein was significantly increased (P ≤ 0.05) in hearts of wild-type mice at 30 min after reperfusion compared with hearts from TLR2D mice. No differences in TNF levels were measured between the two groups after 60 min of reperfusion. Hearts from wild-type mice also showed a trend (P = 0.08) toward higher myocardial IL-1beta protein levels at 30 and 60 min of reperfusion compared with hearts from TLR2D mice. These data suggested that blunted cytokine production contributed, at least in part, to the increased postischemic functional recovery measured in hearts from TLR2D mice.


Figure 3
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  		Fig. 3. Myocardial TNF (A) and IL-1beta (B) protein production after 30 min of zero-flow global ischemia (30I) and 60 min of reperfusion (60R). Values represent means ± SE (n = 6–9/time point). *P ≤ 0.05 WT vs. TLR2D mice.

 
Etanercept improves postischemic functional recovery after ischemia-reperfusion. To determine whether TNF production played a role in the onset of LV dysfunction in our model, hearts isolated from wild-type and TLR2D mice were treated with etanercept before the onset of ischemia, as well as during resuscitation. Etanercept administration resulted in improvement of postischemic LV functional recovery after reperfusion in both wild-type and TLR2D hearts (Fig. 4) compared with untreated mice (Fig. 2A). As shown, there were no differences in %LVDP after etanercept administration.


Figure 4
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  		Fig. 4. Effect of TNF neutralization on myocardial I/R injury. Percent recovery of developed pressure in hearts isolated from WT (n = 5) and TLR2D (n = 5) mice after I/R and treatment with etanercept (protocol as in METHODS).

 
HMGB1 expression is upregulated in the heart after ischemia-reperfusion injury. To determine whether HMGB1 was upregulated in the heart after ischemia-reperfusion injury, the cellular localization of HMGB1 in the heart was assessed by immunostaining normal perfused hearts (80 mmHg; sham-operated hearts) and hearts that underwent ischemia-reperfusion injury. Expression of HMGB1 was noted predominantly in the nucleus of normal perfused wild-type and TLR2D hearts (Fig. 5A). Consistent with reports in liver ischemic injury (38), after ischemia-reperfusion HMGB1 expression was enhanced in cytoplasm of cardiomyocytes from both wild-type and TLR2D hearts (Fig. 5A). Western blot analysis also revealed increased HMGB1 protein expression in the cytoplasmic extracts from both wild-type (2.2-fold) and TLR2D hearts (2.2-fold; Fig. 5B). Although HMGB1 expression was increased in the heart, we were unable to detect increased HMGB1 levels in the perfusates of hearts that underwent ischemia-reperfusion injury (Fig. 5C).


Figure 5
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  		Fig. 5. High-mobility group box 1 (HMGB1) expression is upregulated in hearts after I/R. Hearts from WT and TLR2D mice underwent 30 min of zero-flow global ischemia and 60 min of reperfusion. A: immunostain of HMGB1 from sections of sham-operated hearts from WT and TLR2D mice and hearts subjected to I/R (x20 magnification). Images are representative of heart sections from 3 mice/group. B: Western blot analysis for cellular HMGB1 was performed for protein lysates from sham-operated and I/R hearts from WT and TLR2D mice. Blot shown is representative of 3 independent experiments with similar results. C: perfusates from hearts of TLR2D mice undergoing I/R were subjected to Western blot analysis of HMGB1. Blot shown is representative of 3 experiments with similar results. D: effect of recombinant human HMGB1 (rhHMGB1) on myocyte shortening. Cardiac myocytes were exposed to diluent or HMGB1, and contractility was measured in cells from WT (n = 22 cells) and TLR2D (n = 23 cells) mice as described in METHODS. *P ≤ 0.05 vs. diluent-treated cells; NS, not significant.

 
To determine whether low, undetectable concentrations of HMGB1 might have contributed to the TLR2-mediated LV dysfunction, we asked whether HMGB1 was sufficient to provoke cardiac myocyte dysfunction by exposing isolated murine cardiac myocytes from wild-type and TLR2D mice to rhHMGB1. As shown in Fig. 5D, there was no difference in baseline contractility in myocytes isolated from wild-type or TLR2D mice when they were treated with buffer. Addition of rhHMGB1 (1 µg/ml) induced a significant depression of myocyte contraction shortening in cells isolated from both wild-type and TLR2D mice. Thus, although HMGB1 was sufficient to provoke mycocyte dysfunction, these data suggest that TLR2 is not the cognate receptor for HMGB1 in cardiomyocytes.


   DISCUSSION
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 ABSTRACT
 METHODS
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GRANTS
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In this report, we demonstrate the novel finding that TLR2-TIRAP signaling contributes to the development of ischemia-reperfusion-induced LV dysfunction in the adult heart. Hearts from wild-type mice subjected to ischemia-reperfusion injury exhibited reproducible impaired LV function when compared with normal perfused (80 mmHg) hearts. More importantly, the recovery of postischemic contractile performance (%LVDP) was significantly greater (P ≤ 0.05) in hearts from mice lacking TLR2. Given that these studies were performed in isolated perfused hearts, our results suggest that the LV dysfunction in wild-type mice was due to TLR2-mediated signaling in the heart. We provide further support by showing that the recovery of postischemic contractile performance (%LVDP) was also significantly greater (P ≤ 0.05) in hearts from mice deficient in TIRAP, an adaptor molecule that is essential for TLR2 signaling. Moreover, when TLR4-deficient mice and their appropriate control mice were subjected to ischemia-reperfusion injury, the absence of TLR4 had no significant effect on recovery of postischemic contractile performance, suggesting that TLR4 does not play a major role in mediating LV dysfunction following ischemia-reperfusion injury (data not shown). Although the mechanisms for the difference in LV function are unknown, two lines of evidence suggest that blunted expression of proinflammatory mediators may be responsible, at least in part, for the preserved LV function in hearts from TLR2D mice subjected to ischemia-reperfusion. First, both cardiac TNF and IL-1beta levels were lower in hearts from TLR2D mice after ischemia-reperfusion. Second, administration of etanercept, a selective TNF antagonist, abrogated the LV dysfunction after ischemia-reperfusion injury in wild-type mice. Although etanercept also improved the postischemic LV functional recovery in hearts from TLR2D mice, this effect was less pronounced than in wild-type mice. Taken together, these data indicate that TLR2-mediated signaling contributes to the LV dysfunction following ischemia-reperfusion injury through increased expression of inflammatory mediators. Relevant to our findings, Shishido et al. (35) recently reported that TLR2 signaling also plays a role in cardiac remodeling after myocardial infarction. Survival rates were significantly higher in TLR2D mice than in wild-type mice 4 wk after myocardial infarction, and fractional shortening was preserved at 1 and 4 wk after infarct in TLR2D mice compared with wild-type mice. In addition, myocardial fibrosis in hearts of TLR2D mice was significantly less than in wild-type mice and correlated with reduced transforming growth factor-beta and collagen type 1 mRNA expression.

In mammalian species, there are at least 10 TLRs, and each has a distinct function in innate immune recognition (26). In response to environmental "danger" signals, represented by structural motifs not normally expressed by cells, TLRs can mediate intracellular signals that lead to inflammatory gene expression. Indeed, oxidative stress in neonatal rat cardiomyocytes has been shown to activate NF-{kappa}B via TLR2, suggesting that this receptor may be capable of monitoring cellular injury caused by ischemic stress and therefore may be crucial for the initiation of a proinflammatory innate response (12). Molecules released by stressed cells (heat shock protein 60 and 70) or injured tissue (fibronectin) have been shown to activate TLR2 and TLR4 signaling leading to NF-{kappa}B activation (3, 29, 30). Meng et al. (28) recently demonstrated that TLR4 plays a role in the TNF response and myocardial depression following hemorrhagic shock. However, this study did not determine whether endogenous ligands were released and were responsible for activating the TLR4 signaling during hemorrhagic shock. Recently, HMGB1, a mobile chromatin protein that leaks out from necrotic cells, has been shown to interact in vitro with TLR2 and TLR4 (31, 32). Tsung et al. (38) demonstrated that HMGB1 is an early mediator of injury and inflammation in liver ischemia-reperfusion and have implicated TLR4 as one of the receptors that is involved in the process. It was proposed that released HMGB1 from damaged or necrotic liver cells produced an early inflammatory response by activating the TLR4 pathway. In this study we show for the first time that cardiac HMGB1 expression increases early after reperfusion of the ischemic heart. Consistent with the CK release data suggesting that zero-flow ischemia resulted in similar myocyte injury in both groups, the increase in HMGB1 expression was similar in hearts from wild-type and TLR2D mice (2.2-fold increase). Moreover, our data indicate that the improved recovery of postischemic contractile performance in TLR2D mice is not likely to be due to a diminished ability to respond to HMGB1; that is, rhHMGB1 was able to induce similar degrees of contractile dysfunction in isolated ventricular myocytes from wild-type and TLR2D mice. Thus, although HMGB1 has the potential to adversely affect cardiac myocyte contractility, this effect does not appear to be mediated via TLR2. Although at the present time it is not possible to completely discern the endogenous ligand or ligands released during ischemic injury to the heart, the data presented strongly suggest that TLR2-mediated signaling can directly enhance the expression of genes that play a role in inflammatory responses through activation of the TIRAP pathway.

The findings presented herein are consistent with a growing body of literature that suggests that the heart possesses an intrinsic or "innate stress response" system that is activated following tissue injury, as we and others have suggested (20). The results of our study are intriguing insofar as they suggest that classic pattern recognition receptors that are expressed by the heart, such as TLR2, -3, -4, and -6, are capable of sensing tissue injury or danger signals following ischemia-reperfusion injury. Thus the challenge that lies ahead will be to elucidate the nature of the endogenous danger signals that activate these receptors with the intent of employing this information to reduce tissue injury following ischemia-reperfusion injury. Finally, it will also be important to determine whether there exists a biological link between activation of this innate stress response system and ischemic preconditioning. Further studies need to be conducted to address these interesting possibilities.


   GRANTS
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 ABSTRACT
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 GRANTS
REFERENCES
 
This research was supported by National Institutes of Health Grants HL-58081, HL-42250, HL-073017, and GM-62474.


   ACKNOWLEDGMENTS
 
We thank Ping Yang and Feng Gao for technical assistance. K. J. Tracey serves as consultant for Critical Therapeutics, Inc.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. L. Mann, Winters Center for Heart Failure Research, 1709 Dryden-BCM, 620-Rm 9.83, Houston, TX 77030 (e-mail: dmann{at}bcm.tmc.edu)

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. Section 1734 solely to indicate this fact.

* Y. Sakata and J.-W. Dong contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
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 DISCUSSION
 GRANTS
 REFERENCES
 

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