Extracellular Heat Shock Protein 60, Cardiac Myocytes, and Apoptosis

Abstract

Rationale: Previously, we have found that changes in the location of intracellular heat shock protein (HSP)60 are associated with apoptosis. HSP60 has been reported to be a ligand of Toll-like receptor (TLR)-4. Objective: We hypothesized that extracellular HSP60 (exHSP60) would mediate apoptosis via TLR4. Methods and Results: Adult rat cardiac myocytes were treated with HSP60, either recombinant human or with HSP60 purified from the media of injured rat cardiac myocytes. ExHSP60 induced apoptosis in cardiac myocytes, as detected by increased caspase 3 activity and increased DNA fragmentation. Apoptosis could be reduced by blocking antibodies to TLR4 and by nuclear factor B binding decoys, but not completely inhibited, even though similar treatment blocked lipopolysaccharide-induced apoptosis. Three distinct controls showed no evidence for involvement of a ligand other than exHSP60 in the mediation of apoptosis. Conclusions: This is the first report of HSP60-induced apoptosis via the TLRs. HSP60-mediated activation of TLR4 may be a mechanism of myocyte loss in heart failure, where HSP60 has been detected in the plasma. (Circ Res. 2009;105:1186-1195.)Key Words: Toll-like receptor-4 Ⅲ apoptosis Ⅲ heat shock protein 60 Ⅲ cardiac myocytes Ⅲ tumor necrosis factor Ⅲ TLR4 Ⅲ inflammation T oll-like receptors (TLRs) have been recognized in the last 15 years as an important part of the immune system. The TLRs are a key component of innate immunity, a primitive immunity characterized by the rapid recognition of bacterial and other motifs as dangerous, followed by an inflammatory response that includes the production of cytokines, such as tumor necrosis factor (TNF)-␣. Heat shock protein (HSP)60 is thought to be a ligand of TLR4, which has been found on the surface of cardiac myocytes. 1,2 In the immune system, activation of TLR4 is characterized by activation of nuclear factor (NF)B followed by production of TNF-␣. Limited studies have addressed the function of the TLRs in nonimmune system cells. We hypothesized that extracellular (ex)HSP60 activated TLR4 and that this would induce cardiac myocyte apoptosis.Lipopolysaccharide (LPS) has also been identified as a ligand for TLR4. Some controversy persists as to whether observed effects with other proteins activating TLR4 do so directly, or are actually contaminated with LPS. 3 However, it is becoming clear that extracellular HSPs have an important role in cell signaling. 4 To address the issue of LPS contamination, in addition to careful controls, we examined the effect of LPS on apoptosis, and the effect of a TLR4 blocking antibody on the LPS and exHSP60 induced apoptosis.We report here that exHSP60 binds selectively to the cardiac myocyte and induces apoptosis. Apoptosis is decreased by anti-TLR4 blocking antibodies but not by blocking antibodies to TLR-2 or CD14. These findings imply that HSP60 released during cardiac injury can have a paracrine effect on neighboring myocytes leading to cell death. This is the first report of HSP60 having a toxic effect on cardiac myocyte...

Up‐regulated TLR4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

“…The HSP60 is a DAMP molecule that can be actively secreted from ischaemic cardiomyocytes through specific pathways dependent on both lipid rafts and exosomes 31. Studies from other labs and ours have showed that ischaemia, either alone or followed by reperfusion, induces marked release of HSP60 from cardiomyocytes, which can activate TLR4 and induce cytokine expression in cardiomyocytes 15, 18, 31, 32. Furthermore, we found HSP60 in the circulation of post‐infarct CHF rats 16.…”

Heat Shock Protein-60 and Risk for Cardiovascular Disease

“…It could be postulated that post-translational modifications and/or single nucleotide polymorphisms may be involved. Additionally, elevated HSP60 levels in plasma may stimulate activation of TLR4, which has been shown in adult rats to be responsible for CMC death [38].…”

Inflammatory and fibrotic responses of cardiac fibroblasts to myocardial damage associated molecular patterns (DAMPs)

“…A number of HSPs are also known to act as DAMPs when released from necrotic cells including HSP60, HSP70 and HSP27. The inflammatory response to myocardial ischaemia/reperfusion is mediated in part via cellular release of cardiac HSP60 acting via TLR4 to induce cardiomyocyte apoptosis and cytokine release [51,109], although it is not clear whether CF play a role in this. HSP70 is also released into the circulation early after MI, and extracellular HSP70 stimulated inflammatory cytokine release from monocytes via TLR4 [110].…”