The heat shock proteins (HSP) are a highly conserved family of proteins with critical functions in protein folding, protein trafficking, and cell signaling. These proteins also protect the cell against injury. HSP60 has been found in the extracellular space and has been identified in the plasma of some individuals. HSP60 is thought to be a "danger signal" to the immune system and is also highly immunogenic. Thus extracellular HSP60 is possibly toxic to the cell. The mechanism by which HSP60 is released into the extracellular space is unknown, as is whether it is released by cardiac myocytes. We investigated several different pathways controlling protein release including the classic, Golgi-mediated pathway. We found that HSP60 is released via exosomes, and that within the exosome, HSP60 is tightly attached to the exosome membrane.

Background-Heat shock proteins (HSPs) are well known for their ability to "protect" the structure and function of native macromolecules, particularly as they traffic across membranes. Considering the role of key mitochondrial proteins in apoptosis and the known antiapoptotic effects of HSP27 and HSP72, we postulated that HSP60, primarily a mitochondrial protein, also exerts an antiapoptotic effect. Methods and Results-To test this hypothesis, we used an antisense phosphorothioate oligonucleotide to effect a 50% reduction in the levels of HSP60 in cardiac myocytes, a cell type that has abundant mitochondria. The induced decrease in HSP60 precipitated apoptosis, as manifested by the release of cytochrome c, activation of caspase 3, and induction of DNA fragmentation. Antisense treatment was associated with an increase in bax and a decrease in bcl-2 secondary to increased synthesis of bax and degradation of bcl-2. A control oligonucleotide had no effect on these measurements. We further demonstrated that cytosolic HSP60 forms a macromolecular complex with bax and bak in vitro suggesting that complex formation with HSP60 may block the ability of bax and bak to effect apoptosis in vivo. Lastly, we show that as cytosolic (nonmitochondrial) HSP60 decreases, a small unbound fraction of bax appears and that the amount of bax associated with the mitochondria and cell membranes increases. Conclusions-These results support a key antiapoptotic role for cytosolic HSP60. To our knowledge, this is the first report suggesting that interactions of HSP60 with bax and/or bak regulate apoptosis.

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...

Amione G, Knowlton AA. HSP60 in heart failure: abnormal distribution and role in cardiac myocyte apoptosis. Am J Physiol Heart Circ Physiol 293: H2238-H2247, 2007. First published August 3, 2007; doi:10.1152/ajpheart.00740.2007.-Heat shock protein (HSP) 60 is a mitochondrial and cytosolic protein. Previously, we reported that HSP60 doubled in end-stage heart failure, even though levels of the protective HSP72 were unchanged. Furthermore, we observed that acute injury in adult cardiac myocytes resulted in movement of HSP60 to the plasma membrane. We hypothesized that the inflammatory state of heart failure would cause translocation of HSP60 to the plasma membrane and that this would provide a pathway for cardiac injury. Two models were used to test this hypothesis: 1) a rat model of heart failure and 2) human explanted failing hearts. We found that HSP60 localized to the plasma membrane and was also found in the plasma early in heart failure. Plasma membrane HSP60 localized to lipid rafts and was detectable on the cell surface with the use of both flow cytometry and confocal microscopy. Localization of HSP60 to the cell surface correlated with increased apoptosis. In heart failure, HSP60 is in the plasma membrane fraction, on the cell surface, and in the plasma. Membrane HSP60 correlated with increased apoptosis. Release of HSP60 may activate the innate immune system, promoting a proinflammatory state, including an increase in TNF-␣. Thus abnormal trafficking of HSP60 to the cell surface may be an early trigger for myocyte loss and the progression of heart failure.heat shock proteins; protein trafficking; plasma membrane; cytokines HSP60 IS AN IMPORTANT MEMBER of the heat shock protein family, thought to be primarily a mitochondrial protein, although it is encoded by the nuclear genome. Previously, our laboratory (8) reported that HSP60 is doubled in end-stage heart failure. HSP60 binds Bax and Bak in the cytosol of the myocyte, and reduction in HSP60 precipitates apoptosis (6). Simulated ischemia in cardiac myocytes resulted in translocation of HSP60 to the plasma membrane before reoxygenation (5). Redistribution of HSP60 to the plasma membrane was associated with movement of Bax to the mitochondria and apoptosis.Observations of HSP60 translocation in isolated myocytes motivated studies on the redistribution of HSP60 in the failing heart, where apoptosis is thought to be a mechanism of myocyte death. To address this question, we developed a rat model of heart failure with ligation of the left anterior descending artery. Cardiac echo was used to follow changes in function and chamber size. Studies were conducted long after coronary ligation and thus reflected the progressive changes of the failing ventricle, rather than ischemia. Upregulation of HSP60 correlated with increased expression of proinflammatory cytokines, brain natriuretic peptide (BNP), and atrial natriuretic peptide (ANP) 9 and 12 wk postligation. In addition, we examined distribution of HSP60 in explanted human hearts with dilated (DCM) and ischem...

A nonsignificant 6.3% absolute reduction in the restenosis rate and a nonsignificant difference in MLD were observed in this study. Nonetheless, we still cannot rule out a beneficial effect of IVUS guidance, although this may have gone undetected owing to a lack of statistical power. A significant increase was observed in immediate and 6-month lumen size, as detected by IVUS, indicating that ultrasound guidance in stent deployment may be beneficial.