We found that the constitutively expressed HSC70 is released into the coronary effluent during global ischemia-reperfusion, and we detected HSC70 in the extracellular space of the myocardium after global ischemia-reperfusion. Therefore HSC70 escapes from cardiac cells during ischemia-reperfusion. Further, the HSC70 release we detected was from isolated, buffer-perfused hearts, which excludes any contribution from other organs or blood cells. Thus our results demonstrate that myocardial ischemia-reperfusion causes HSC70 to be released from the heart itself.
Several studies have demonstrated the release of HSC70 in vitro from various cell types. HSC70 is released from glia cells (30
) and from K562 erythroleukemic cells treated with IFN-γ, IL-10, or hyperthermia (3
). In addition, Saito et al. (23
) found that HSPs including HSC70 are released from injured cells. The present study is the first to demonstrate the release of HSC70 from the heart. The HSC70 release we observed is likely due to cell injury, but active secretion of HSC70 from cells has also been reported (3
). Further study is needed to determine what mechanism(s) and which cell type(s) are involved in myocardial HSC70 release. To our knowledge, no in vivo clinical or animal studies have examined myocardial HSC70 release or measured circulating HSC70 levels. Several studies have reported that the inducible HSP70 is released into the circulation after stressful or injurious events including myocardial infarction (9
) and cardiac surgery with obligatory global myocardial ischemia-reperfusion (10
). These studies do not, however, address whether HSC70 is also released. Since the constitutive HSC70 is much more abundant in most cells, including cardiac myocytes, than the inducible HSP70, it is possible that HSC70 release after myocardial infarction and cardiac surgery with global ischemia-reperfusion has even greater significance than HSP70 release. Further studies are needed to examine HSC70 release in those clinical situations.
In this study, we found that HSC70-specific antibody improves cardiac functional recovery after global ischemia-reperfusion and reduces expression of the proinflammatory cytokines TNF-α, IL-1β, and IL-6. We also found that recombinant HSC70 depresses cardiac contractility and increases the expression of TNF-α, IL-1β, and IL-6. Therefore, extracellular HSC70 appears to play a role in cardiac dysfunction and in the expression of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 after global ischemia-reperfusion. It is well-known that TNF-α, IL-1β, and IL-6 are cardiodepressant. In an earlier study (5
), we linked TNF-α and IL-1β to reduced cardiac contractility after ischemia-reperfusion. Since extracellular HSC70 plays a critical role in myocardial expression of cardiodepressant cytokines, the release of HSC70 from the myocardium may play a role in the cardiac dysfunction and inflammatory response observed in vivo after cardiac surgery with obligatory global myocardial ischemia-reperfusion.
In our previous study (5
), we found that TLR4 plays a critical role in postischemic myocardial cytokine production and cardiac dysfunction. However, how myocardial TLR4 activation occurs during ischemia-reperfusion remains unknown. In this study, after finding that extracellular HSC70 contributes to the inflammatory response and myocardial functional injury after ischemia-reperfusion, we then examined whether extracellular HSC70 exerts these effects through TLR4. We found that HSC70 activates the p38 MAPK and NF-κB pathways; induces myocardial expression of TNF-α, IL-1β, and IL-6; and depresses cardiac contractility in TLR4-competent hearts but not in TLR4-defective hearts. HSC70 also induced a TNF-α response in macrophages from TLR4-competent mice but not from TLR4-defective mice. These results provide direct evidence that extracellular HSC70 activates NF-κB and p38 MAPK; induces the myocardial expression of TNF-α, IL-1β, and IL-6; and depresses cardiac contractility via a TLR4-dependent mechanism. Thus extracellular HSC70 appears to be one of the factors that activate myocardial TLR4.
In this study, we found that HSC70 induces p38MAPK and NF-κB activation in a TLR4-dependent manner and that p38MAPK and NF-κB activation correlates with the expression of proinflammatory cytokines. It seems that the p38MAPK and NF-κB pathways are involved in signaling mechanisms in the HSC70-induced, TLR4-dependent inflammatory response. TLR4 signaling involves both MyD88-dependent and MyD88-independent pathways (15
). In the MyD88-dependent pathway, MyD88 recruits IRAK4, which then phosphorylates IRAK1 to propagate the proinflammatory signal, leading to the phosphorylation of the IKK complex and MAPKs, including p38 MAPK (14
). In the MyD88-independent pathway, TLR4 utilizes TRIF-related adaptor molecules to activate NF-κB. Previous studies (2
) have shown that HSP70 activates TLR4 and induces proinflammatory cytokine production in monocytes via the MyD88/IRAK/NF-κB signaling pathway. Future studies are required to determine the relative role of the MyD88-dependent and -independent pathways in the activation of NF-κB and p38 MAPK by HSC70.
It has been suggested that activation of TLR4 by recombinant proteins is partly due to endotoxin contamination (32
). Although we used a preparation of rHSC70 with very little endotoxin contamination (<5.0 pg/µg protein by Limulus assay), we examined whether that amount of endotoxin in our rHSC70 preparation contributed to the inflammatory response by treating macrophages with rHSC70 in the presence of the endotoxin antagonist polymyxin B (5.0 µg/ml). We found that the endotoxin antagonist had a minimal effect on rHSC70-induced TNF-α production. Thus endotoxin is not responsible for the proinflammatory effect of rHSC70. In addition, the recombinant HSC70 fragment, which also contains a trace amount of endotoxin, did not induce a cytokine response in the heart. These results support the conclusion that the induction of a TLR4-dependent proinflammatory response by HSC70 is due to the native properties of the protein.
HSC70 is a constitutively expressed cytoplasmic protein that functions intracellularly primarily as a molecular chaperone (36
). It has two main functional domains, an ATPase or NBD and an SBD (20
). Deletion studies (7
) have shown that these two domains are sufficient for HSC70 to function as a chaperone in clathrin uncoating. We tested whether an HSC70 fragment without the SBD induces cytokine production. In our isolated heart experiments, the HSC70 fragment did not induce cytokine expression nor did it influence cardiac contractility. Since the proinflammatory and cardiodepressive effects of HSC70 require the SBD, it is most likely the SBD of HSC70 that interacts with and/or activates TLR4. We applied FRET analysis to macrophages incubated with HSC70 to examine whether HSC70, when applied extracellularly, interacts with TLR4. The results showed increased cell-surface HSC70 levels and increased FRET signals between HSC70 and TLR4. Therefore, it appears that extracellular HSC70 interacts with TLR4 to activate that receptor.
In summary, this study demonstrates for the first time that 1) the myocardium releases HSC70 during global ischemia-reperfusion, 2) extracellular HSC70 contributes to the postischemic myocardial inflammatory response and to cardiac dysfunction, 3) HSC70 applied extracellularly is sufficient to induce the myocardial inflammatory response and cardiac functional changes through a TLR4-dependent mechanism, 4) the substrate-binding domain of HSC70 is required to induce these TLR4-dependent effects, and 5) extracellular HSC70 interacts with TLR4. These findings indicate that extracellular HSC70 depresses cardiac function by modulating the myocardial innate immune response and suggest that extracellular HSC70 may represent a novel target for preserving cardiac function during global ischemia-reperfusion.