Myocardial Ischemia Activates an Injurious Innate Immune Signaling via Cardiac Heat Shock Protein 60 and Toll-like Receptor 4*

Innate immune response after transient ischemia is the most common cause of myocardial inflammation and may contribute to injury, yet the detailed signaling mechanisms leading to such a response are not well understood. Herein we tested the hypothesis that myocardial ischemia activates interleukin receptor-associated kinase-1 (IRAK-1), a kinase critical for the innate immune signaling such as that of Toll-like receptors (TLRs), via a mechanism that involves heat shock proteins (HSPs) and TLRs. Coronary artery occlusion induced a rapid myocardial IRAK-1 activation within 30 min in wild-type (WT), TLR2−/−, or Trif−/− mice, but not in TLR4def or MyD88−/− mice. HSP60 protein was markedly increased in serum or in perfusate of isolated heart following ischemia/reperfusion (I/R). In vitro, recombinant HSP60 induced IRAK-1 activation in cells derived from WT, TLR2−/−, or Trif−/− mice, but not from TLR4def or MyD88−/− mice. Both myocardial ischemia- and HSP60-induced IRAK-1 activation was abolished by anti-HSP60 antibody. Moreover, HSP60 treatment of cardiomyocytes (CMs) led to marked activation of caspase-8 and -3, but not -9. Expression of dominant-negative mutant of Fas-associated death domain protein or a caspase-8 inhibitor completely blocked HSP60-induced caspase-8 activation, suggesting that HSP60 likely activates an apoptotic program via the death-receptor pathway. In vivo, I/R-induced myocardial apoptosis and cytokine expression were significantly attenuated in TLR4def mice or in WT mice treated with anti-HSP60 antibody compared with WT controls. Taken together, the current study demonstrates that myocardial ischemia activates an innate immune signaling via HSP60 and TLR4, which plays an important role in mediating apoptosis and inflammation during I/R.

Innate immune response after transient ischemia is the most common cause of myocardial inflammation and may contribute to injury, yet the detailed signaling mechanisms leading to such a response are not well understood. Herein we tested the hypothesis that myocardial ischemia activates interleukin receptor-associated kinase-1 (IRAK-1), a kinase critical for the innate immune signaling such as that of Toll-like receptors (TLRs), via a mechanism that involves heat shock proteins (HSPs) and TLRs. Coronary artery occlusion induced a rapid myocardial IRAK-1 activation within 30 min in wild-type (WT), TLR2 ؊/؊ , or Trif ؊/؊ mice, but not in TLR4 def or MyD88 ؊/؊ mice. HSP60 protein was markedly increased in serum or in perfusate of isolated heart following ischemia/reperfusion (I/R). In vitro, recombinant HSP60 induced IRAK-1 activation in cells derived from WT, TLR2 ؊/؊ , or Trif ؊/؊ mice, but not from TLR4 def or MyD88 ؊/؊ mice. Both myocardial ischemia-and HSP60-induced IRAK-1 activation was abolished by anti-HSP60 antibody. Moreover, HSP60 treatment of cardiomyocytes (CMs) led to marked activation of caspase-8 and -3, but not -9. Expression of dominant-negative mutant of Fas-associated death domain protein or a caspase-8 inhibitor completely blocked HSP60-induced caspase-8 activation, suggesting that HSP60 likely activates an apoptotic program via the death-receptor pathway. In vivo, I/R-induced myocardial apoptosis and cytokine expression were significantly attenuated in TLR4 def mice or in WT mice treated with anti-HSP60 antibody compared with WT controls. Taken together, the current study demonstrates that myocardial ischemia activates an innate immune signaling via HSP60 and TLR4, which plays an important role in mediating apoptosis and inflammation during I/R.
Toll-like receptors (TLRs) 2 represent the first line of defense against invading microbial pathogens and play a critical role in both innate and adaptive immunity (1,2). TLRs recognize invading pathogens through molecular pattern recognition, transduce signals via distinct intracellular pathways involving a unique set of adaptor proteins such as myeloid differentiation primary-response gene 88 (MyD88) and Toll/interleukin-1 receptor domain-containing adaptor protein inducing interferon-␤ (Trif), and kinases such as interleukin (IL) receptor-associated kinase-1 (IRAK-1), and ultimately lead to activation of transcription factors and inflammatory responses. All TLRs, except TLR3, signal through the common MyD88-dependent pathway (3). TLR3 exclusively and TLR4 partially signal via the MyD88-independent but Trif-dependent pathway (4). In addition to their critical roles in the host defense against invading pathogens, accumulative evidence suggests that TLRs can also recognize endogenous ligands produced by stressed tissues (5) and play an important role in "non-infectious" tissue injury (6 -10). For example, in isolated cells, studies have demonstrated that TLR4 recognizes heat shock proteins (HSPs) (11)(12)(13)(14), fibrinogen (15), and soluble heparan sulfate (16) and modulates cell inflammation and survival. However, the critical role of these endogenous TLR ligands and their downstream signaling under in vivo pathological conditions, such as ischemic myocardial injury, is unclear.
Our previous studies have shown that myocardial IRAK-1, the kinase critical for innate immune signaling, quickly becomes activated in response to transient ischemia (9). However, the signaling mechanisms leading to and the biological significance of the ischemia-induced myocardial IRAK-1 activation are unknown. The present study was designed 1) to determine whether or not TLRs are responsible for the ischemia-induced IRAK-1 activation, 2) to identify one or more endogenous ligands for TLR signaling during myocardial ischemia, and 3) to determine the role of these endogenous ligands in myocardial inflammation and apoptosis during ischemia/reperfusion (I/R).
Animals-C57BL/6J, C57BL/10ScSn, and TLR4 def mice (C57BL/10ScCr) were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/10ScCr is also referred to as C57BL/ 10ScNJ (stock no. 003752) with wild-type (WT) Il12rb2 allele. C57BL/10ScCr mice have a deletion of the Tlr4 gene, which results in the absence of both mRNA and protein and thus in a defective response to LPS. Tlr4 lps-del differs from the Tlr4 Lps-d mutation of C3H/HeJ mice, a point mutation of Tlr4 gene that causes an amino acid substitution (17). C57BL/10ScSn (WT/ B10) mice were used as the appropriate WT controls for the TLR4 def mice, whereas C57BL/6J (WT/B6) mice were used as the controls for all other knock-out mice. TLR2 Ϫ/Ϫ mice were generated by Takeuchi et al. (18). MyD88 Ϫ/Ϫ mice were generated by Kawai and colleagues (19) and had been backcrossed for Ͼ 10 generations into the C57BL/6J strain. Trif Ϫ/Ϫ mice were generated by Yamamoto et al. (4). All mice used in the study were 8 -12 weeks old, male, and weighed between 20 and 30 g. All animal protocols used in the study were approved by the Subcommittee on Research Animal Care of the Massachusetts General Hospital (Boston, MA).
Mouse Models of Myocardial Ischemia and Sample Preparation-The models have been described previously (20,21). Briefly, mice were anesthetized with ketamine (120 mg/kg) and xylazine (4 mg/kg), intubated, and ventilated in a volumecontrol mode. Mouse body temperature was maintained within normal limit (36.5-37.5°C). A left thoracotomy was performed, and the left anterior descending coronary artery was ligated under a surgical microscope. For I/R, the left anterior descending artery ligature was released after the indicated time of occlusion, and reperfusion was visually confirmed. For the ischemia model, a permanent tie was used. In the sham-operated animals, a suture was passed under the left anterior descending artery but not tied. To harvest serum and the hearts, animals were euthanized. Clotted blood in Eppendorf tubes was centrifuged at 3,000 rpm for 10 min, and serum was frozen for HSP60 ELISA. The hearts were removed, and the ventricular chambers were opened, washed three times with cold PBS, frozen in liquid nitrogen, and stored in a Ϫ80°C freezer for subsequent IRAK kinase assay, HSP60 ELISA, cytokine qRT-PCR, and caspase-3 Western blotting.
Ex Vivo Annexin V Imaging-Fluorescence reflectance imaging of cell death was performed using a conjugate of annexin V and a near infrared fluorochrome (Annexin-Vivo-750 (AV-750), PerkinElmer Life Sciences). The fluorescent annexin construct was similar to the MRI-detectable annexin constructs, which we have used to detect apoptosis in the myocardium (22,23). However, a fluorescent moiety rather than a magnetic nanoparticle was used to create the imaging readout. Mice were injected intravenously with 100 l of AV-750 at the onset of reperfusion and euthanized for imaging 4 h later. Each heart was sectioned into 1-mm-thick slices and imaged in fluorescence reflectance mode on an IVIS Spectrum imaging system (Caliper Life Sciences, Hopkinton, MA). AV-750 accumulation was imaged with an excitation wavelength of 745 nm, an emission wavelength of 800 nm, and a 10-s exposure time. Immediately prior to sacrifice, the coronary artery was re-ligated, and fluorescent microspheres (Invitrogen, Carlsbad, CA) were injected to demarcate the area-at-risk (AAR). Microsphere distribution and AV-750 accumulation were imaged simultaneously on the IVIS Spectrum system. An excitation wavelength of 540 nm, a 600 nm emission filter, and a 60-s exposure were used to image the microspheres. All images were acquired with a spatial resolution of 135 m. The AAR in a slice was defined by the absence of fluorescent microspheres, and the AV-750positive area was defined by a signal Ͼ2 standard deviations above background. The percentage of the AAR with annexin accumulation was compared in the two groups of mice (control IgG versus anti-HSP60) with a Mann-Whitney test.
Ex Vivo Model of Myocardial I/R-The ex vivo model has been described previously (20,24). Briefly, mice were heparinized and euthanized. The hearts were excised, and aortae were cannulated and retrograde perfused at a constant rate (3 ml/min) with modified Krebs-Henseleit buffer equilibrated with 95% O 2 -5% CO 2 at a pH of 7.4 and at 37°C. After 10 min of control perfusion in a Langendorff perfusion apparatus, the hearts were exposed to 30 min of no-flow global ischemia followed by 30 min of reperfusion. Perfusates were harvested for 10 s at 0, 5, 10, 20, and 30 min after reperfusion started, stored, and used for subsequent HSP60 ELISA.
Cell Cultures-Rat neonatal CMs were prepared as described previously with minor modifications (9). Briefly, the hearts were isolated, dissected from major vessels, and cut into small pieces. The heart tissues were then incubated in ADS buffer (pH 7.35, 116 mM NaCl, 20 mM HEPES, 0.8 mM Na 2 HPO 4 , 5.6 mM glucose, 5.4 mM KCl, and 0.8 mM MgSO 4 ) containing 0.4 mg/ml collagenase 2 and 0.6 mg/ml pancreatin (Worthington, Lakewood, NJ) at 37°C for 5 min in a shaker. Cell suspension was slowly removed, and the remaining myocardial tissues were further incubated with the enzyme buffer for four more times. Cells in suspension were collected, spun, and resuspended in DMEM containing 20% FBS and 4.5% D-glucose. Fibroblasts were removed by plating cells on 10-cm dishes for 60 min. Neonatal CMs were then plated in 6-well plate (3 ϫ 10 5 cells/well) and incubated in CO 2 incubator at 37°C for 5 days before experiments were performed.
Bone marrow cells were harvested from the tibias and femurs of mice, cultured, and differentiated into macrophages in the presence of macrophage colony-stimulating factor as described previously with minor modifications (21). Briefly, the cells were resuspended at 4 ϫ 10 6 /10 ml in RPMI 1640 medium supplemented with 10 ng/ml macrophage colony-stimulating factor, 10% FBS, and 5% horse serum. Four days later, culture media were changed, and macrophages were ready for experiments at day 7.
Treatments of Cell Cultures-Cultured beating neonatal CMs or macrophages were washed twice with pre-warm serum-free RPMI 1640 medium containing 0.05% BSA. The cell cultures were then treated in DMEM supplemented with 10% FBS. For additional control experiments, HSP60 or LPS was used in combination with 50 g/ml PMB or heated at 95°C for 10 min. For in vitro HSP60 blocking experiments, HSP60 protein was incubated with an HSP60 blocking antibody at a molar ratio of 1:3 at 4°C for 30 min before applied to cell cultures.
Adenovirus-mediated Transgene Expression-The recombinant adenoviral vectors (Ad.) that encode GFP (Ad.GFP), WT IRAK-1, kinase-deficient mutant IRAK-1 (K239S), WT Fas-associated death-domain protein (FADD), or the DED-deleted dominant-negative mutant of FADD (FADD-DN) were constructed as described previously (26). Each viral vector contains a CMV-driven double transcription cassette for both target protein and the control GFP. All Ad constructs have been made using the pAd Easy system and have been characterized previously (9,27). WT Ad contamination was excluded by the absence of PCR-detectable E1 sequences. Cultured CMs were incubated with the above adenoviral vectors overnight. The transgene expression was confirmed by GFP as well as IRAK-1 or FADD protein expression as determined by immunoblotting (26).
RNA Extraction and Quantitative RT-PCR Analysis-Total RNAs were extracted from mouse myocardial tissues or rat neonatal CMs using TRIzol reagent, and cDNAs were synthesized by reverse-transcriptase reaction. cDNA gene sequences complementary to 18 S rRNA and target RNA were amplified by PCR and quantitated using a Mastercycler ep realplex realtime PCR system (Eppendorf, Hauppauge, NY). Sequences of primers can be supplied at request. Changes in relative gene expression normalized to 18 S rRNA levels were determined using the relative threshold cycle method as described previously (20).
Immunoprecipitation, Western Blotting, and IRAK-1 Kinase Assay-The experiments were performed as described previously (9). Briefly, CMs (3 ϫ 10 6 cells) in a 6-well plate were scraped, spun down, and washed once with 5 ml of cold PBS. Freshly frozen mouse myocardial tissues were prepared into powder. Cell pellets or tissue powders were dissolved in 1 ml of cold Nonidet P-40 lysis buffer (pH 7.4, 10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40), plus protease inhibitors (1 g/ml leupeptin, 1 g/ml aprotinin, and 1 g/ml pepstatin A), and incubated at 4°C for 60 min. Cell and tissue lysates were then centrifuged at 14,000 rpm for 20 min, and supernatants were saved for immunoprecipitation (9). For immunoprecipitation, 2.5 g of polyclonal anti-IRAK-1 antibody was added to cell lysates containing equal amounts of proteins and incubated at 4°C overnight on a rotator. To harvest the IRAK-1-antibody complex, 60 l of pre-washed 50% slurry protein G (catalogue no. 17-0618-01, GE Healthcare, Piscataway, NJ) was added, and the mixture was incubated at 4°C for 2 h, spun down, and washed four times with cold lysis buffer. A fraction of the protein G beads (1/4) was used for IRAK-1 Western blotting as described previously (9). The remaining beads were washed twice and incubated in 110 l of kinase assay buffer (pH 7.6, 20 mM HEPES, 20 mM MgCl 2 , 20 mM ␤-glycerophosphate, 20 mM p-nitrophenylphosphate, 1 mM EDTA, 1 mM NaVO 3 , and 1 mM benzamidine). To start the IRAK-1 kinase assay, 5 M cold ATP, 10 Ci of [␥-32 P]ATP, and 2 g of myelin basic protein were added to 110 l of immunoprecipitates, and the mixture was incubated at 37°C for 30 min. At the end of the assay, immunoprecipitates were mixed with SDS sample buffer, heated, and separated in 4 -20% gradient SDS-PAGE. The 32 P-labeled myelin basic protein was visualized by autoradiography for indications of IRAK-1 activity.
HSP60 ELISA-Freshly frozen mouse myocardial tissues were ground into powder. Tissue powders were homogenized on ice using Nonidet P-40 lysis buffer. Lysates were centrifuged at 14,000 rpm for 15 min, and supernatants were stored and used for subsequent HSP60 ELISA. Serum and perfusates were harvested as described above. HSP60 levels in heart, serum, or perfusates were measured using an HSP60 ELISA kit.
Statistics-Unless stated otherwise, all data are expressed as the mean Ϯ S.E. of at least three independent experiments and were analyzed with a two-tailed, unpaired Student's t test. The null hypothesis was rejected for p Ͻ 0.05.

RESULTS
Myocardial Ischemia-induced IRAK-1 Activation Is TLR4and MyD88-dependent-WT mice were subjected to coronary artery ligation or sham procedure for 15 min to 2 h, and myocardial IRAK-1 activity was examined. As indicated in Fig. 1A, transient myocardial ischemia induced a rapid increase in IRAK-1 activity as evidenced by enhanced phosphorylation of myelin basic protein. The peak effect appeared to be at 30 min after the onset of ischemia. To determine the role of TLRs in mediating the ischemia-induced IRAK-1 activation, we subjected mice deficient in TLR4 (TLR4 def ) or TLR2 (TLR2 Ϫ/Ϫ ) to the same ischemia protocol. Transient myocardial ischemia failed to induce IRAK-1 activation in TLR4 def mice (Fig. 1B), but was capable to do so in TLR2 Ϫ/Ϫ at a similar level as WT (Fig. 1C). MyD88 and Trif are two distinct adaptors critical for TLR4 signaling, but MyD88 is responsible for recruiting and activating IRAK-1 upon TLR4 stimulation. To determine whether or not the two adaptor signaling pathways control the ischemia-induced, TLR4-mediated myocardial IRAK-1 activation, we examined myocardial IRAK-1 kinase activity in MyD88 Ϫ/Ϫ and Trif Ϫ/Ϫ mice subjected to the transient ischemia. As demonstrated in Fig. 1D, MyD88 Ϫ/Ϫ mice failed to show any IRAK-1 activation following transient myocardial ischemia, whereas Trif Ϫ/Ϫ mice responded normally.
HSP60 Is Released from Ischemic Myocardium-We postulated that an endogenous ligand(s) acted on TLR4 and triggered the rapid myocardial IRAK-1 activation during ischemia. To test the hypothesis, we first examined the mRNA levels of several stress proteins reportedly to work through TLR4, including HSP20, HSP60, HSP70, biglycan, high mobility group box protein, and hyaluronan in myocardium. We found that none of the ligand mRNA tested was significantly altered 30 min after myocardial ischemia (supplemental Fig. S1). We reasoned that the proposed endogenous ligands might be released from ischemic myocardium instead of de novo synthesized during the short period of ischemia. HSP60 has been described as a TLR4 ligand (13,29,30) and is produced in cultured adult CMs upon hypoxia-reoxygenation (12). To test whether or not HSP60 is released during myocardial ischemia, we subjected WT and TLR4 def mice to coronary artery ligation for 15 or 30 min followed by 30 min of reperfusion. There was a significant decrease in the myocardial HSP60 levels upon myocardial I/R in both WT and TLR4 def mice ( Fig. 2A) and a corresponding increase (Ͼ10-fold) in the serum HSP60 (Fig. 2B) compared with their sham-operated controls. To determine the cardiac origin of the serum HSP60 after I/R, we isolated and subjected mouse hearts to a global I/R in a Langendorff perfusion apparatus. As shown in Fig. 2C, there was a significant decrease in HSP60 levels in the heart after 30 min of ischemia and 30 min of reperfusion compared with the perfused hearts. Importantly, there was a significant amount of HSP60 released into the perfusates at 10 min of the reperfusion in the isolated heart (Fig.  2D). Together, these data suggest that transient ischemia leads to a rapid release of myocardial HSP60 and that the I/R-induced HSP60 release does not involve TLR4 signaling.
HSP60 Induces IRAK-1 Activation in CMs-To elucidate the role of HSP60 in IRAK-1 activation, we treated cultured CMs with recombinant human HSP60 for 60 min and tested IRAK-1 activity. As indicated in Fig. 3A, like TLR2 (P3C) and TLR4 (LPS) ligands, HSP60 at 1 g/ml induced a robust IRAK-1 activation. In contrast, the TLR3 ligand, I:C (25 g/ml), which signals via Trif, did not induce IRAK-1 activation in CMs. The effect of HSP60 was dose-dependent (Fig. 3B). Because the human HSP60 used was a recombinant protein produced in an E. coli expression system, the HSP60 preparation might contain a low level of endotoxin (31)(32)(33). Using a Limulus amebocyte lysate assay, we determined the endotoxin concentration in the HSP preparation (Enzo Life) to be 20 pg/mg. The calculated  final concentration of endotoxin in the HSP60-treated cultures was extremely low (0.02 pg/ml). Nevertheless, we performed a series of control studies to rule out any potential effect of the low level of LPS in the HSP60-induced IRAK-1 activation. PMB is a cationic cyclic polypeptide that acts primarily by binding membrane phospholipids and disrupting the cytoplasmic membrane, inducing pore formation in bacterial cell walls. PMB binds to the lipid-A portion of the LPS in the cell membrane of Gram-negative bacteria and, thus, is a specific LPS antagonist (34). We found that PMB completely blocked LPSinduced IRAK-1 activation but had no impact on the effect of HSP60 (Fig. 3C). Conversely, pretreatment of HSP60 with heat at 95°C for 10 min abolished the ability of HSP60 to induce IRAK-1 activation but had no impact on the effect of LPS (Fig.  3D). Moreover, pretreatment of HSP60 with the specific anti-HSP60 antibody (35) completely blocked the HSP60-, but not the LPS-, induced IRAK-1 activation (Fig. 3E). Importantly, to determine whether HSP60 is responsible for myocardial IRAK-1 activation in vivo following ischemia, we treated mice with the anti-HSP60 antibody or control IgG 5 min prior to the onset of myocardial ischemia. As shown in Fig. 4, prior administration of the blocking antibody significantly attenuated the myocardial IRAK-1 activation. Taken together, these data strongly suggest that recombinant HSP60 is capable of activating IRAK-1 signaling in CMs in vitro and that endogenously released HSP60 is at least in part responsible for IRAK-1 activation in ischemic myocardium in vivo.
HSP60 Induces CM Apoptosis via the DR Pathway-CM apoptosis occurs during transient myocardial ischemia and may contribute to I/R injury (36 -39). To test if HSP60 activates apoptosis signaling in CMs, we treated rat neonatal CMs with HSP60 in DMEM containing serum and glucose and examined DNA fragmentation 24 h and caspase activities 6 h later. SD/hypoxia was employed as a positive control for apoptosis as we have previously established that SD/hypoxia induces a rapid activation of caspases and CM apoptosis (10,26). As shown in Fig. 6A, both HSP60 and SD/hypoxia induced a significant amount of DNA fragmentation. SD/hypoxia also induced activation of all three caspases tested, i.e. caspase-3, -8, and -9. HSP60, on the other hand, induced activation of only caspase-3 and -8, but not -9 (Fig. 6B). Caspase-8 is the key enzyme for the death receptor (DR) pathway of cellular apoptosis, whereas caspase-9 is essential for the mitochondrial pathway (40 -42). To further probe the role of the DR signaling pathway in the HSP60-elicited CM apoptosis, we tested the effect of FADD, a DR adaptor known for its critical role in caspase-8 recruitment and activation during the DR-mediated apoptosis (26, 43) on  the HSP60-induced caspase-8 activation. We have previously shown that overexpression of FADD is sufficient to induce caspase-8 activation in CMs, whereas expression of a truncation mutant that lacks the death effector domain and possesses dominant negative effect (FADD-DN) is protective against SD/hypoxia-induced caspase-8 activation (26). As illustrated in Fig. 6C, HSP60-or Ad-mediated transgene expression of FADD-WT resulted in a significant increase in caspase-8 activity in CMs. Combination of HSP60 and Ad.FADD-WT induced additional caspase-8 activation. Importantly, either Ad-mediated expression of FADD-DN (Fig. 6C) or z-IETD-fmk (a casepase-8 inhibitor) (Fig. 6D) effectively blocked the HSP60induced caspase-8 activation. As expected, z-IETD-fmk also blocked SD/hypoxia-induced caspase-8 activation (Fig. 6D). Furthermore, HSP60-induced CM apoptosis was dose-dependent (Fig. 6, E and F) and inhibited by the anti-HSP60 antibody (Fig. 6, G and H) as evidenced by DNA fragmentation ELISA and caspase-3 activity. Taken together, this series of data suggests that HSP60 likely induces CM apoptosis via the FADD/ caspase-8-dependent DR pathway.
TLR4 Deletion or HSP60 Blocking Antibody Attenuates I/Rinduced Myocardial Apoptosis in Vivo-To determine the role of HSP60 3 TLR4 signaling in I/R-induced myocardial apoptosis, we subjected WT and TLR4 def mice to 30 min of ischemia and 4 h of reperfusion. Myocardial apoptosis was assessed by caspase-3 cleavage and ex vivo fluorescent annexin-V imaging. As illustrated in Fig. 8A, myocardial I/R induced an increase in cleaved caspase-3 expression in WT mice. The cleaved caspase-3 was significantly lower in TLR4 def mice. In addition, myocardial I/R induced a marked increase in AV-750 accumu-FIGURE 6. HSP60 induces CM apoptosis via DR pathway. A, apoptosis assayed by DNA fragmentation. Beating CMs were either incubated with (HSP60) or without (Con) HSP60 (1 g/ml) in medium containing 10% of FBS at 37°C and 5% CO 2 /95% air or subjected to SD/hypoxia for 24 h. *, p Ͻ 0.05 versus Con. B, HSP60-induced activation of caspase-3 and -8, but not -9. CMs were treated as above, except the treatment time was 6 h. *, p Ͻ 0.05; **, p Ͻ 0.01 versus Con. lation within the AAR, representing a significant level of cell death in the ischemic myocardium (Fig. 8B). Importantly, AV-750 accumulation was lower in TLR4 def mice than WT mice. To elucidate the role of endogenous HSP60 in the I/Rinduced myocardial apoptosis, we treated WT mice with the anti-HSP60 blocking antibody or control IgG (5 g, intravenously, twice, 5 min prior to ischemia and 5 min prior to reperfusion). As illustrated in Fig. 9, the anti-HSP60 blocking antibody led to a significant decrease in the I/R-induced caspase-3 cleavage (Fig. 9A). Again, AV-750 accumulation was seen in the majority of the AAR. In mice injected with a control antibody, 96.5 Ϯ 1.1% of the total AAR was annexin-positive (n ϭ 5). A small but statistically significant reduction in the annexin uptake was seen in mice injected with the HSP60 blocking antibody (n ϭ 6) (90.3 Ϯ 2.9%, p Ͻ 0.05) (Fig. 9B).
TLR4 Deletion or Anti-HSP60 Antibody Attenuates I/Rinduced Myocardial Cytokine Expression-As shown in Fig.  10, 30 min of ischemia and 4 h of reperfusion induced a marked increase in the myocardial gene expression of IL-1␤, IL-6, KC, MIP-2, and MCP-1. TLR4 genetic deletion (Fig.  10A) or prior administration of the specific anti-HSP60 antibody (Fig. 10B) markedly attenuated the I/R-induced cytokine expression compared with WT or IgG controls, respectively.

DISCUSSION
Using the genetically modified mouse model, the current study demonstrated that ischemia-induced activation of IRAK-1, a kinase critical for innate immune signaling in the heart, was dependent on TLR4-MyD88 signaling. Transient ischemia led to a rapid release of myocardial HSP60 as demonstrated in both in vivo and ex vivo models of I/R. Several lines of evidence suggest that the endogenously released HSP60 mediates ischemia-induced IRAK-1 activation via the TLR4-MyD88 pathway. First, recombinant HSP60 was capable of inducing IRAK-1 activation in cultured CMs. Second, similar to ischemia-induced IRAK1 activation in vivo, the HSP60-induced IRAK-1 activation was also TLR4-and MyD88-dependent. Third, neutralizing HSP60 antibody markedly reduced IRAK-1 activation induced by myocardial ischemia in vivo and by HSP60 in vitro. We found that HSP60 activated an apoptotic program likely through the FADD/caspase-8-dependent DR signaling pathway in CMs. Finally, we demonstrated that both  TLR4 deficiency and the anti-HSP60 antibody significantly attenuated I/R-induced caspase-3 activation, myocardial cell death, and multiple cytokine expression in vivo. Thus, the current study demonstrates that myocardial ischemia activates an innate immune signaling by releasing HSP60 and through TLR4-MyD88. The HSP60 3 TLR4 signaling proves to be critical for myocardial apoptosis and inflammation during I/R (Fig. 11).
In addition to acting as a sensor to invading foreign pathogens and playing a critical role in host innate immunity (1, 2), accumulative evidence from various animal models indicates that TLRs may respond to endogenous tissue effectors produced under stressful and injurious conditions and contribute to pathological processes (5,8). Such examples include traumainduced hepatic injury (44), ventilation-induced lung injury (45), brain injury (46), and ischemic myocardial injury (6,7,47). In the heart, for example, systemic deficiency of TLRs such as TLR2 (47) and TLR4 (6, 7) results in reduced infarct sizes and improved function compared with WT mice after transient ischemia. However, the molecular mechanisms by which TLRs contribute to I/R injury are not well understood. It has been proposed that TLR signaling may contribute to I/R injury by mediating acute myocardial inflammatory response, because attenuation of myocardial inflammation is often found in ischemic myocardium in mice deficient of TLRs and MyD88 (7,20,48). Studies in chimeric models further demonstrate that MyD88 signaling may contribute to I/R injury by mediating neutrophil recruitment and migratory functions (21). The current study provides the detailed signaling mechanisms by which myocardial innate immune system is activated and contributes to myocardial apoptosis and inflammation in response to ischemic stress.
We chose IRAK-1 activity as the main endpoint in the study to assess TLR innate immune signaling because of its upstream location and critical function in the TLR signaling. Although other kinases or transcription factors such IKK␤, I-B, or NFB are important and could be tested, these downstream effectors are not specific for TLR signaling. Several other receptor systems such as TNF␣, the IL-6 family, endothelin-1, and cardiotropin can also activate these kinases and NFB-dependent pathways (49). MyD88 is a key adaptor protein that is critical for transducing signals from IL-1 receptor family members and all TLR family members (3,50), except TLR3. MyD88 signals via IRAK-1 and other downstream kinases, including IKK␤ and IB, eventually leading to the activation of NFB and inflammatory cytokine production (51). On the other hand, Trif is a key adaptor protein responsible for TLR3 and TLR4 signaling pathways that respond to viral and bacterial stimulation and result in the production of type I interferons (52). Stimulation of the type I interferon pathways leads to induction of a specific set of genes, including chemokines (e.g. CXCL10) (53), and antimicrobial/anti-viral response genes (54). Because Trif is not essential for IRAK-1 activation, the lack of impact of Trif deficiency on ischemia-induced IRAK-1 activation is not surprising, but it highlights the specific contribution of MyD88 signaling to this process (Fig. 11).
HSPs are highly conserved molecules that fulfill a range of functions, such as immunomodulation, intracellular assembly, folding, and translocation of oligomeric proteins (55), and have been implicated as stress proteins in response to myocardial ischemic injuries (56 -60). Cardiac surgeries with obligatory myocardial ischemia, such as coronary artery bypass grafting, were found to cause the release of HSP60 (61,62). HSP60 is released from injured CNS (13), from rat adult CMs subjected to transient hypoxia in vitro (12), and from failing heart (59). Our study demonstrates that transient myocardial ischemia leads to acute release of endogenous HSP60 from the heart. This is supported by several observations. First, in vivo, 15-30 min of left anterior descending artery ligation resulted in a decrease in the myocardium and a corresponding increase in the serum in HSP60 levels. Second, isolated perfused heart released HSP60 to the perfusate after I/R. The ex vivo study confirmed the cardiac origin of HSP60 released during I/R. Finally, qRT-PCR analysis of many major endogenous TLR ligands, including HSP60, failed to show any transcript increase following 30 min of myocardial ischemia. This suggests that the released HSP60 from ischemic myocardium during reperfusion is not due to de novo synthesis but rather from intracellular storage of cardiac cells.
To determine whether or not HSP60 is the endogenous ligand responsible for IRAK-1 activation during I/R, we tested the ability of HSP60 to induce IRAK-1 activation in isolated CMs. The in vitro data demonstrate that recombinant HSP60 is capable of inducing a robust IRAK-1 activation. Several lines of evidence in the study indicate that this effect is specific for HSP60 and not due to potential endotoxin contamination in the HSP60 preparation. There was only a minute concentration of LPS in the HSP60 preparation by Limulus testing. The final endotoxin concentration in cell cultures was extremely low (Ͻ0.02 pg/ml). PMB, an endotoxin neutralizer, completely abolished LPS-induced IRAK-1 activation and had no effect on the ability of HSP60-induced IRAK-1 activation. In contrast, FIGURE 11. Schematic view of the proposed HSP60-TLR4 signaling in response to myocardial ischemia. In response to ischemic stress, myocardium releases the stress protein HSP60 locally and into the systemic circulation. The endogenously released HSP60 acts on the cell surface TLR4 in paracrine and/or autocrine fashion. HSP60 activates myocardial IRAK-1 via TLR4-MyD88 signal pathway. HSP60 also induces CM apoptosis via FADDand caspase-8-dependent mechanisms as both FADD-DN and z-IETD-fmk can block the HSP60-induced apoptosis. Finally, I/R-induced myocardial apoptosis and inflammation (cytokine expression) in vivo are partially dependent on HSP60-TLR4 signaling. TLR4 deletion or anti-HSP60 antibody attenuates I/R-induced myocardial apoptosis and inflammation.
heating denatured HSP60 and thus led to inactivation of the protein but had no effect on the ability of LPS to activate IRAK-1. HSP60-induced IRAK-1 activation was dose-dependent and completely blocked by its neutralizing antibody. Finally, the blocking experiments using the HSP60 antibody in vivo prior to coronary artery ligation demonstrated that endogenous HSP60 was responsible for the ischemia-induced IRAK-1 activation. To establish the link between HSP60 and TLRs, we tested the ability of HSP60 to induce IRAK-1 activation in macrophages harvested from WT mice and mice deficient in TLR2, TLR4, MyD88, or Trif. These loss-of-function studies clearly indicate that, similar to ischemia-induced IRAK-1 activation in vivo, HSP60-induced IRAK-1 activation depends on TLR4-MyD88 signaling. Of note, whereas TLR2 stimulation (by P3C) is sufficient to activate IRAK-1 activation in cultured cells in vitro, TLR2 is apparently not required for the ischemiainduced myocardial IRAK-1 activation in vivo.
HSP60 has been known for its ability to induce cellular apoptosis in isolated CMs and in microglia. In rat adult CMs, Kim et al. have shown that HSP60 induces apoptosis partially via TLR4-and NFB-dependent mechanisms (12). In CNS, HSP60 induces apoptosis via the TLR4-MyD88 signaling pathway (13). We extend these previous studies and identify that HSP60 induces CM apoptosis via the caspase-8/FADD-mediated signaling pathway. HSP60 specifically activates caspase-8, the enzyme critical for the DR-induced apoptosis, and shows no apparent effect on caspase-9, the enzyme essential for the mitochondrial pathway. On the other hand, as we have previously reported (26), SD/hypoxia induces apoptosis by activating both caspase-8 and -9. HSP60-induced caspase-8 activation can be blocked by FADD-DN and by z-IETD-fmk, whereas overexpression of FADD-WT is sufficient to induce caspase-8 activation. These data suggest that both caspase-8 and FADD are essential for HSP60-induced CM apoptosis. Intriguingly, LPS, the pathogenic TLR4 ligand, has been reported for its antiapoptotic effect in CMs. CMs pretreated with LPS become more resistant to apoptosis in an in vitro SD/hypoxia model (9). Mice administered with a small dose of LPS 12 h prior to the onset of myocardial I/R are protected with reduced myocardial apoptosis and myocardial infarct sizes (24,63,64). In the presence of serum, unlike HSP60, LPS did not induce apoptosis (data not shown). The reasons for the difference between LPS and HSP60 are unclear and now being investigated.
To elucidate the role of IRAK-1 in apoptosis, we expressed WT or the KD mutant of IRAK-1 in CMs using E1-deleted recombinant adenoviral vector-mediated gene transfer (9,26,65). Again, in the presence of serum, Ad-mediated expression of IRAK-1-WT but not its KD mutant led to significant CM apoptosis as demonstrated by DNA fragmentation and activation of caspase-3, -8, and -9. These data indicate that IRAK-1 expression is sufficient to activate both DR and mitochondrial apoptosis pathways and this pro-apoptotic effect is dependent on its kinase activity. Interestingly, in Ad-IRAK-1-transduced cells, addition of HSP60 led to an additional activity of caspase-3 and -8, but not -9. These data suggest that both HSP60 and IRAK-1 induce a potent apoptotic program in CMs. The data cannot, however, determine if IRAK-1 is required for HSP60-induced apoptosis, and the two activators seem to act somewhat differently, because IRAK-1 expression activates both DR and mitochondrial pathways, whereas HSP60 only activates the DR pathway.
To determine the functional significance of the HSP60driven TLR4 signaling in the pathogenesis of ischemic myocardial injury, we tested whether or not in vivo administration of anti-HSP60 blocking antibody or genetic TLR4 deletion has any impact on the I/R-induced myocardial apoptosis and cytokine production, because both apoptosis and inflammation are known contributors to myocardial I/R injury. We found that anti-HSP60 blocking antibody or TLR4 deletion significantly, but not completely, attenuated I/R-induced caspase-3 cleavage and annexin V accumulation. These data suggest that both HSP60 and TLR4 contribute to caspase activation and cell death during myocardial I/R. Moreover, the finding that TLR4 deletion and HSP60 blocking markedly reduced multiple cytokine gene expression in vivo supports the notion that the HSP60 3 TLR4 signaling plays an essential role in myocardial inflammation during I/R.
In summary, the present study demonstrated that transient myocardial ischemia led to a rapid activation of IRAK-1 through TLR4-MyD88 signaling. We identified that HSP60 was rapidly released from myocardium in response to transient ischemia. HSP60 was capable of inducing IRAK-1 activation in CMs via TLR4-MyD88 signaling and responsible for IRAK-1 activation after ischemia in vivo. HSP60 treatment in CMs induces apoptosis specifically via the DR pathway. We demonstrated that HSP60-TLR4 signaling was important for myocardial apoptosis and inflammation during I/R. Thus, our study reveals a novel signaling mechanism that controls innate immune response during myocardial I/R that may have a deleterious effect on the heart.