Myocardial ischemia induces a rapid activation of innate immune signaling via cardiac heat‐shock protein‐60 and Toll‐like receptor 4

Purpose Toll‐like receptor 4 (TLR4) and its signaling molecule interleukin‐1 receptor‐associated kinase‐1 (IRAK‐1) are important components of innate immune signaling. Our previous studies have demonstrated that transient myocardial ischemia activates IRAK‐1 in cardiomyocytes (CMs). Here we test the hypothesis that myocardial ischemia activates IRAK‐1 signaling by releasing endogenous heat shock proteins (HSPs) and through specific TLR signaling pathways. Methods In vivo and ex vivo ischemic injury; neonatal rat CMs and mouse macrophages;IRAK‐1 Kinase Assay; ELISA. Results Myocardial ischemia induced a time‐dependent increase in myocardial IRAK‐1 activity in WT mice that peaked at 30 min. The activation of IRAK‐1 sustained for a period of 120 min. To determine the upstream event leading to the IRAK‐1 activation following ischemia, we subjected TLR2−/−, TLR4−/−, MyD88−/− or Trif−/− mice to the ischemic injury for 30 min. Similar to WT mice, TLR2−/− mice exhibited a robust increase in myocardial IRAK‐1 activity. In contrast, TLR4−/− and MyD88−/− mice failed to show any IRAK‐1 activation, whereas Trif−/− mice maintained intact in IRAK‐1 activity. Moreover, we found that cardiac HSP60 was released to serum in vivo or to perfusates ex vivo following ischemia. Then we treated CMs and macrophages with recombinant human HSP60. HSP60 activated IRAK‐1 in a dose‐dependent manner, which was abolished by HSP60 blocking antibody or heating, but not by LPS neutralizer. Finally, we found that HSP60 induced IRAK‐1 activation only in cells isolated from WT, TLR2−/− mice, but not from TLR4−/− or MyD88−/− mice. Taken together, these data demonstrate that myocardial ischemia activates IRAK‐1 signaling via endogenously released HSP60 and in a TLR4‐MyD88‐dependent manner. Funding: National Institutes of Health grant GM‐080906.