Myocardial ischemia activates an injurious innate immune signaling via cardiac heat shock protein 60 and Toll-like receptor 4

Abstract

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.

FIGURE 1.

Myocardial ischemia-induced IRAK-1 activation is TLR4- and MyD88-dependent. WT or the genetically modified mice (TLR4^def, TLR2^−/−, Trif^−/−, and MyD88^−/−) were subjected to coronary artery ligation or sham procedures for the indicated periods. Myocardial IRAK-1 activity was assayed by myelin basic protein (MBP) phosphorylation as described under “Experimental Procedures.” Each data point represents mean ± S.E. of three to four individual experiments. A fraction of IRAK-1 immunoprecipitates was used for IRAK-1 kinase assay (upper panel, 3/4) and IRAK-1 immunoblotting (lower panel, 1/4). A, myocardial ischemia by coronary ligation led to rapid activation of IRAK-1 in WT mice. *, p < 0.05 versus sham; B, myocardial ischemia-induced IRAK-1 activation is TLR4-dependent. *, p < 0.05 versus TLR4^def mice; C, myocardial ischemia-induced IRAK-1 activation is TLR2-independent. D, Myocardial ischemia-induced IRAK-1 activation is MyD88-dependent but Trif-independent. *, p < 0.05 versus MyD88^−/− mice.

FIGURE 2.

HSP60 is released from ischemic myocardium. A and B, HSP60 is released from ischemic myocardium in both WT and TLR4^def mice in vivo. WT/B10 and TLR4^def mice were subjected to the sham procedures or coronary artery ligation for 15 or 30 min and then 30 min of reperfusion. At the end of the protocol, myocardium (A) and serum (B) were collected and assayed for HSP60 levels. * and ^#, p < 0.05, ** and ^##, p < 0.01 versus the sham controls. C and D, HSP60 is released from ischemic myocardium of the isolated heart. WT mice were euthanized, and the hearts were perfused in a Langendorff apparatus. After 30 min of perfusion, the hearts were subjected 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 the onset of reperfusion. At the end of perfusion, the hearts were removed and assayed for HSP60. **, p < 0.01 versus the perfused hearts.

FIGURE 3.

Recombinant HSP60 induces IRAK-1 activation in rat neonatal CMs. A, IRAK-1 activation induced by HSP60 and TLR ligands. Beating CMs were incubated with HSP60 (1 μg/ml, lane 2), P3C (1 μg/ml, lane 3), I:C (25 μg/ml, lane 4), and LPS (500 ng/ml, lane 5) at 37 °C for 60 min in DMEM containing 10% FBS. Control cells were treated with PBS (Con, lane 1). B, dose dependence of HSP60-induced IRAK-1 activation. CMs were treated with HSP60 at different concentrations of 0.025 μg/ml (lane 2), 0.1 μg/ml (lane 3), 1 μg/ml (lane 4), and 5 μg/ml (lane 5) at 37 °C for 60 min. Controls were treated with PBS. C, effect of PMB on LPS- and HSP60-induced IRAK-1 activation. HSP60 (1 μg/ml) and LPS (500 ng/ml) were preincubated with or without polymyxin B sulfate (PMB, 50 μg/ml) in 4 °C for 60 min before applied to CMs. CM cultures were treated with LPS without the PMB preincubation (lane 2), LPS with PMB preincubation (lane 3), HSP60 without PMB (lane 4), HSP60 with PMB (lane 5) at 37 °C for 60 min. Controls were treated with PBS (lane 1). D, effect of heat on the HSP60-induced IRAK-1 activation. HSP60 (1 μg/ml) and LPS (500 ng/ml) were heated at 95 °C for 10 min before being applied to CMs. CMs were then treated with LPS without heat (lane 2), LPS with heat (lane 3), HSP60 without heat (lane 4), and HSP60 with heat (lane 5) at 37 °C for 60 min. Controls were treated with PBS (lane 1). E, effect of anti-HSP60 antibody on the HSP60-induced IRAK-1 activation. HSP60 (1 μg/ml) and LPS (500 ng/ml) were preincubated with or without mouse anti-HSP60 antibody (3 μg/ml) at 4 °C for 30 min before being applied to CM cultures. CM cultures were treated with LPS without anti-HSP60 (lane 2), LPS with anti-HSP60 (lane 3), HSP60 without anti-HSP60 (lane 4), or HSP60 with anti-HSP60 (lane 5) at 37 °C for 60 min. Controls were treated with IgG (3 μg/ml, lane 1). Each data point represents mean ± S.E. of four to six individual experiments. **, p < 0.01 versus control.

FIGURE 4.

Myocardial ischemia-induced IRAK-1 activation is blocked by anti-HSP60 antibody in vivo. Control IgG (5 μg) or anti-HSP60 antibody (5 μg) was administered to mice via tail vein 5 min before the onset of myocardial ischemia (30 min, I-30′). Coronary artery was ligated for 30 min. Sham mice received IgG. *, p < 0.05 versus sham + IgG; ^#, p < 0.05 versus I-30′ + IgG.

FIGURE 5.

HSP60-induced IRAK-1 activation is dependent on TLR4-MyD88 signaling. Macrophage cultures were treated with HSP60 (1 μg/ml, lane 2), P3C (1 μg/ml, lane 3), I:C (25 μg/ml, lane 4), or LPS (500 ng/ml, lane 5) at 37 °C for 60 min in DMEM containing 10% FBS. Control cells were treated with PBS (lane 1). Cells were harvested and assayed for IRAK-1 activity as described under “Experimental Procedures.” A, effect of TLR4 deletion on IRAK-1 activation induced by HSP60, P3C, I:C, or LPS. B, effect of TLR2 deletion on IRAK-1 activation induced by HSP60, P3C, I:C, or LPS. C, effect of Trif and MyD88 deletion on IRAK-1 activation induced by HSP60, P3C, I:C, or LPS. Each data point represents mean ± S.E. of four to six individual experiments. A and B, *, p < 0.05 versus control/WT; ^§, p < 0.05 versus HSP60/WT; ^#, p < 0.05 versus P3C/WT; ^Φ, p < 0.05 versus LPS/WT. C, *, p < 0.05 versus control/Trif^−/−; ^§, p < 0.05 versus HSP60/Trif^−/−; ^#, p < 0.05 versus P3C/Trif^−/−; ^Φ, p < 0.05 versus LPS/Trif^−/−.

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 μg/ml) in medium containing 10% of FBS at 37 °C and 5% CO₂/95% air or subjected to SD/hypoxia for 24 h. *, p < 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 < 0.05; **, p < 0.01 versus Con. C, effect of Ad.FADD-WT and -DN on HSP60-induced caspase-8 activation in CMs. CMs were transduced with the Ad viral constructs overnight. The expression levels were confirmed (supplemental Fig. S2). The infected cells were then treated with HSP60 (1 μg/ml) or PBS for 6 h. *, p < 0.05 versus Ad.GFP/PBS; ^§, p < 0.01 versus Ad.FADD-WT/PBS; ^#, p < 0.05 versus Ad.FADD-WT/HSP60. D, effect of z-IETD-fmk on HSP60- and SD/hypoxia-induced caspase-8 activation. Cells were treated with z-IETD-fmk (50 μm) or vehicle for 1 h prior to HSP60 treatment or SD/hypoxia. Six hours later, caspases-8 activity was assayed. *, p < 0.05 versus vehicle/control; ^§, p < 0.01 versus vehicle/HSP60; ^#, p < 0.05 versus vehicle/SD/hypoxia. E and F, dose dependence of HSP60-induced DNA fragmentation and caspase-3 activation. CMs were treated with different doses of HSP60 as indicated at 37 °C for 24 h. Control cells were treated with PBS. *, p < 0.05; **, p < 0.01 versus the control. G and H, effect of anti-HSP60 antibody on HSP60-induced DNA fragmentation and caspase-3 activity. HSP60 (1 μg/ml) were preincubated with mouse anti-HSP60 antibody (3 μg/ml) or control IgG (3 μg/ml) at 4 °C for 30 min before being applied to CMs. CMs were then treated with HSP60 at 37 °C for 24 h. Control cells were treated with IgG (3 μg/ml). *, p < 0.05 versus the control; **, p < 0.01 versus the control. For all above experiments, each data point represents mean ± S.E. of three to four individual experiments.

FIGURE 7.

HSP60 and IRAK-1 induce apoptosis in CMs. Beating CMs were infected with the indicated adenoviral vectors overnight. CMs were then treated with PBS or HSP60 (1 μg/ml) in serum-containing medium. At the end of treatment, CMs were assayed for DNA fragmentation (A) or caspase activities (B–D) as indicated. Each data point represents mean ± S.E. of three to four individual experiments. *, p < 0.05 versus Ad.GFP/PBS; ^§, p < 0.05 versus Ad.IRAK-1-WT/PBS; ^#, p < 0.05 versus Ad.IRAK-1-KD/PBS. ^φ, p < 0.05 versus Ad.IRAK-1-WT/PBS.

FIGURE 8.

Impact of TLR4 deletion on I/R-induced caspase-3 cleavage and cell death. WT/B10 and TLR4^def mice were subjected to coronary artery ligation for 30 min followed by 4 h of reperfusion. A, caspase-3 Western blot. Each data point represents mean ± S.E., n = 3. *, p < 0.05 versus sham; ^#, p < 0.05 versus WT-I/R. B, ex vivo fluorescent annexin imaging of cell death. Fluorescent Annexin-Vivo-750 (AV) and microspheres were administered as described under “Experimental Procedures.” Each heart was cut from the apex to base into five slices (top to bottom). Areas devoid of red fluorescent microspheres were considered as area-at-risk (AAR).

FIGURE 9.

Anti-HSP60 antibody attenuates I/R-induced myocardial caspase-3 cleavage and cell death. Mice were administered with control IgG or anti-HSP60 (5 μg) prior to myocardial ischemia and again to reperfusion as described under “Experimental Procedures.” A, caspase-3 Western blot. Each data point represents mean ± S.E., n = 3. *, p < 0.05 versus sham + IgG; ^#, p < 0.05 versus I/R + IgG. Ex vivo fluorescent annexin imaging of cell death is shown. Fluorescent Annexin-Vivo-750 (AV) and microspheres were administered as described under “Experimental Procedures.” Each heart was cut from the apex to base into five slices (top to bottom). Areas devoid of red fluorescent microspheres were considered as area-at-risk (AAR).

FIGURE 10.

TLR4 deletion or anti-HSP60 antibody markedly attenuates ischemia-induced myocardial cytokine expression. A, WT/B10 and TLR4^def mice were subjected to coronary artery ligation for 30 min and then 4 h of reperfusion. At the end of protocol, myocardium was assayed for cytokine mRNA expression (TNFα, IL-1β, IL-6, IL-10, KC, MCP-1, MIP-2, and ICAM-1) as described under “Experimental Procedures.” *, p < 0.05 versus WT/sham; ^#, p < 0.05 versus WT/I/R. B, WT mice were administered with control IgG or anti-HSP60 (5 μg) prior to myocardial ischemia and again to reperfusion as described under “Experimental Procedures.” *, p < 0.05 versus sham + IgG; ^#, p < 0.05 versus I/R + IgG, n = 5.

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 FADD- and 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.