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...

Heat shock proteins (HSPs) are a cardioprotective class of proteins induced by stress and regulated by the transcription factor, heat shock factor (HSF)-1. 17β-estradiol (E(2)) indirectly regulates HSP expression through rapid activation of nuclear factor-κB (NF-κB) and HSF-1 and protects against hypoxia. As males experience a loss of protective cellular responses in aging, we hypothesized that aged menopausal (old ovariectomized) rats would have an impaired HSP response, which could be prevented by immediate in vivo E(2) replacement. After measuring cardiac function in vivo, cardiac myocytes were isolated from ovariectomized adult and old rats with and without 9 weeks of E(2) replacement. Myocytes were treated with E(2) in vitro and analyzed for activation of NF-κB, HSF-1, and HSP expression. In addition, we measured inflammatory cytokine expression and susceptibility to hypoxia/reoxygenation injury. Cardiac contractility was reduced in old ovariectomized rats and could prevented by immediate E(2) replacement in vivo. Subsequent investigations in isolated cardiac myocytes found that in vitro E(2) activated NF-κB, HSF-1, and increased HSP 72 expression in adult but not old rats. In response to hypoxia/reoxygenation, myocytes from adult, but not old, rats had increased HSP 72 expression. In addition, expression of the inflammatory cytokines TNF-α and IL-1β, as well as oxidative stress, were increased in myocytes from old ovariectomized rats; only the change in cytokine expression could be attenuated by in vivo E(2) replacement. This study demonstrates that while aging in female rats led to a loss of the cardioprotective HSP response, E(2) retains its protective cellular properties.

BackgoundIt has been reported that Toll-like receptor 4 (TLR4) deficiency reduces infarct size after myocardial ischemia/reperfusion (MI/R). However, measurement of MI/R injury was limited and did not include cardiac function. In a chronic closed-chest model we assessed whether cardiac function is preserved in TLR4-deficient mice (C3H/HeJ) following MI/R, and whether myocardial and systemic cytokine expression differed compared to wild type (WT).ResultsInfarct size (IS) in C3H/HeJ assessed by TTC staining after 60 min ischemia and 24h reperfusion was significantly smaller than in WT. Despite a smaller infarct size, echocardiography showed no functional difference between C3H/HeJ and WT. Left-ventricular developed pressure measured with a left-ventricular catheter was lower in C3H/HeJ (63.0 ± 4.2 mmHg vs. 77.9 ± 1.7 mmHg in WT, p < 0.05). Serum cytokine levels and myocardial IL-6 were higher in WT than in C3H/HeJ (p < 0.05). C3H/HeJ MI/R showed increased myocardial IL-1β and IL-6 expression compared to their respective shams (p < 0.05), indicating TLR4-independent cytokine activation due to MI/R.ConclusionThese results demonstrate that, although a mutant TLR4 signaling cascade reduces myocardial IS and serum cytokine levels, it does not preserve myocardial function. The change in inflammatory response, secondary to a non-functional TLR-4 receptor, may contribute to the observed dichotomy between infarct size and function in the TLR-4 mutant mouse.

The induction of the heat shock response is accepted to be a protective response, reducing injury and improving cell survival. However, when inflammation precedes heat shock there is an unexpected increase in injury, known as the heat shock paradox, which is hypothesized to be a mechanism underlying multi-organ dysfunction. We hypothesized that the heat shock paradox would occur in adult cardiac myocytes and that heat shock factor (HSF)1 would contribute to injury. Heat shock (HS) at 42°C and TNF (10 ng/ml) were used as the HS and the inflammatory insult, respectively. The combination of TNF followed by HS (TNF/HS) caused the greatest amount of apoptosis in adult rat cardiac myocytes. TNF/HS resulted in an increase in heat shock protein (HSP) 60, compared to untreated cells, those receiving HS/TNF, or TNF alone. There was no increase in heme oxygenase 1 in any of the groups. HSP72 increased in all the groups, with the greatest levels with TNF/HS. NFκB activation was greatest with TNF/HS. Pretreatment with a DNA binding decoy for HSF1 prevented the increase in HSPs and decreased apoptosis in all groups. However, the increase in iNOS, seen in all treatment groups, was unaffected by the HSF1 binding decoy. We conclude that the heat shock paradox occurs in adult cardiac myocytes, that HSP60 is increased as part of the heat shock paradox, and that HSF1 activation contributes to injury.

Heat Shock Proteins (HSPs) are intracellular molecular chaperones, which preserve protein folding and protect cells from injury.

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